Photo-acoustic detection device and method

DESCRIPTION

Device and method of photoacoustic detection

Technical field

The field of the invention is medical tests. One aspect of the invention relates to devices and a method of detecting analytes in a body fluid sample, an example of which is circulating tumor cells in a blood sample.

Background Technique

The detection of analytes in body fluid samples is widely used for medical and other purposes. Applications include the detection of pathogens, proteins or other chemical compounds in blood, urine, bile, saliva or other body fluids. Some exemplary applications include drug detection, disease detection, detection of a particular protein and the like. By way of a particular example, the detection of circulating tumor cells (CTC) in human blood and lymphatic systems has the potential to help in making clinical decisions in the treatment of cancer. The presence of CTC can mean the appearance of metastasis, indicate a relapse or can be used to monitor the progression of a disease.

Current techniques and devices for the detection of CTCs have limits. Polymerase chain reaction with reverse transcription (RT-PCR) is a technique that is used in practice. This technique involves RNA analysis. The technique takes significant time and involves several stages that require the expertise of technicians to perform tests. With the requirement of such expertise the potential for technical error appears. Freezing, cultivation, testing, etc., in the RT-PCR technique take as much time as expertise. In addition, the results are not immediately available to a doctor in charge.

Laser flow cytometna also has the potential to analyze samples for CTC. However, CTC detection is an evolving research area and optimal detection techniques remain a work in progress. However, when perfected, and if it is, the technique will continue to be one that shares part of the inconveniences of RT-PCR; for example, the involvement of expert technicians and delays in obtaining and interpreting the results.

Powerful diagnostic instruments allow a fast and accurate evaluation. Critical for the treatment is the initial stage of detection of the appearance of metastasis or recapda, and the monitoring of the progress of the disease and the response of the disease to a course or treatment that is being applied. Having accurate information about a metastasis can provide a physician in charge with the opportunity to be more effective and to address the particular phase of the disease indicated by metastasis. Accurately and quickly detecting the presence of CTCs has the potential to advance the state of diagnosis and treatment of cancer.

MacKenzie et al., "A laser photoacoustic sensor for analyte detection in aqueous systems", Sensors and Actuators B, 11 (1993) 213-220 describe the absorption of laser pulses by a liquid, whereby the absorbed energy is converted into heat and a localized thermal expansion occurs that causes a pressure wave at ultrasonic frequencies that can be detected with a suitable transducer.

Gulshan Ara et al., "Irradiation of pigmented melanoma cells with high intensity pulsed radiation generates acoustic waves and kills cells," Lasers in surgery and medicine 10 (1990) No. 1, New York, describe that a visible short-pulse radiation generates acoustic waves by thermal expansion.

Laufer et al., "Pulsed near-infrared photoacoustic spectroscopy of blood", Proceedings of SPIE, vol. 5320, 2004, pp.

57-68 describe the determination of oxygen saturation of a hematfes saline suspension in vitro by directing laser pulses to a cuvette containing the hematfes suspension and detecting the resulting acoustic signal.

EP 0369 176 A2 describes the labeling of an antigen-antibody complex with a coloring material and the determination of the photoacoustic properties of the brand by photoacoustic spectroscopy.

EP 0256474 A2 describes the use of photoacoustic spectrometry to determine the concentration of a particulate substance in a sample. In the method, an excitation beam is directed to a cell that contains a liquid sample comprising a particulate substance that absorbs the excitation light and generates a photoacoustic signal whose magnitude is measured and used to determine the concentration of the particulate substance in the sample.

Disclosure of the invention

An exemplary method for detecting an analyte in a body fluid sample comprises the steps of exposing the body fluid sample to electromagnetic energy to cause thermoelastic expansion in the analyte, and detecting a photoacoustic signal in the sample that is the result of the thermoelastic expansion, as defined in claim 1. An exemplary system for the detection of one or more analytes in a body fluid comprises a test chamber having at least one side wall configured to contain a sample of testing of a body fluid, a source of electromagnetic energy configured to direct a source of enema into said test chamber through said at least one side wall and induce a thermoelectric expansion in the one or more analytes, and a sensor configured to detect thermoelastic expansion in the test sample, as defined in claim 12.

An additional exemplary apparatus is a photoacoustic metastasis detection device comprising a container for containing a sample of fluid to be tested for the presence or absence of circulating tumor cells, a laser for subjecting the sample of fluid to a light that induces a photoacoustic reaction in circulating tumor cells or in a marker attached thereto, and an acoustic sensor to detect an induced photoacoustic reaction in the sample of fluid contained in said container.

Brief description of the drawings

FIG. 1 is a flow chart illustrating an exemplary method of the invention;

FIG. 2 is a flow chart illustrating a second exemplary method of the invention;

FIG. 3 is a schematic illustration of an exemplary system of the invention and is also useful for illustrating an exemplary method of the invention;

FIG. 4 illustrates the test cell of the system of FIG. 3, illustrating FIG. 4 (B) the cell when viewed along line 4B-4B of FIG. 4 (A) in the direction shown;

FIG. 5 illustrates the test cell of the system of FIG. 3;

FIG. 6 schematically illustrates the detection of a thermoelastic event using the system sensor of FIG.

3;

FIG. 7 illustrates data resulting from the practice of an exemplary system and method of the invention, FIG. 7 (A) data for a test sample that includes live melanoma and presenting FIG. 7 (B) data for a test sample that includes RPMI culture broth and no melanoma;

FIG. 8 is a schematic illustration of an exemplary alternative flow cell; Y

FIG. 9 is a schematic illustration of the flow cell of FIG. 8 seen along line 9-9 of FIG. 8 in the direction shown.

Best mode for carrying out the invention

Before presenting exemplary embodiments of the invention, it will be appreciated that the present invention includes methods, as well as systems. For example, a method of the invention may include stages of use of a system of the invention, and a system of the invention, when used, may be useful for performing stages of a method of the invention. Accordingly, it will be appreciated that, when describing a method of the invention, a system description can also be provided. In addition, when a system of the invention is described, a description of a method of the invention can be made. For example, it will be appreciated that, when describing details of a system of the invention, a description of one or more methods of the invention is provided simultaneously, and vice versa.

The present invention is directed to methods and systems for detecting an analyte in a sample of body fluid. As used herein, it is intended that the term "analyte" be interpreted broadly in the sense of being a chemical, biological or other substance, or a material of interest. By way of example, an analyte can be a protein, a pathogen, such as one or more cancerous tumor cells, a chemical compound or the like. In addition, as used herein, it is intended that the term "body fluid" be interpreted broadly with the meaning of a liquid sample that contains some fluid that originated in a body. Examples include samples of body fluids containing blood, blood plasma, urine, bile, saliva, semen, sperm, breast milk, cerebrospinal fluid, intracellular fluid and the like. It is important to note that, as used herein, the expression "body fluid sample" is not limited to the fluid obtained from the body only, but is also intended to include samples prepared using the fluid and a diluent fluid or vehicular fluid . By way of example, the expression "body fluid sample" encompasses a sample prepared by suspending a body fluid such as leukocytes in a saline solution.

To optimally describe various exemplary embodiments of the invention, a description of general embodiments (and, accordingly, a general apparatus) is initially provided. FIG. 1 is a flow chart illustrating an exemplary method of the invention. A sample of body fluid is exposed to a source of electromagnetic energy to cause thermoelastic expansion in the analyte contained in the sample. Block 10 The body fluid sample may be any of the particular samples expressed above, including, for example, urine, blood or saliva. In addition, it can be, for example, a test sample that includes one of these diluted, suspended or other materials present together with a vehicular fluid. A vehicular fluid may be a solvent or diluent fluid, including examples aqueous solutions, a saline solution and the like.

The source of electromagnetic energy is a laser light. A thermoelastic expansion occurs when a material absorbs energy, heats and expands accordingly. The result is a photoacoustic signal.

The method of FIG. 1 includes, then, a step of using an acusti sensor

resulting from thermoelastic expansion. Block 12. The photoacoustic signal can be a wave or other signal. The detection may include, for example, the use of a suitable detector to detect an acoustic wave that travels through the test sample. An example includes detecting the deflection of a diaphragm in fluid contact with the solution when the acoustic wave makes contact with the diaphragm of a sensor. Another is to use an optical detector to measure a signal such as a disturbance (which may be a refractive change) in the sample after thermoelastic expansion.

By way of an additional example, FIG. 2 presents an additional exemplary method of the invention. This exemplary embodiment is directed to a method of detecting a pathogen, such as a cancerous tumor cell, in a blood sample. However, the particular application of the analyte for this exemplary embodiment could be easily changed.

In an initial stage, a blood sample is obtained from a patient. Block 20. The sample can be, for example, of a volume of only a fraction of a milliliter to a liter or more. It can be obtained through a puncture of the skin with a needle, intravenously or similar. The leukocytes are then separated from the blood sample. Block 22. This can be done, for example, using a centrifuge or other known method of separation. The amount of leukocytes separated will depend, at least to some extent, on the size of the initial blood sample.

Leukocytes thus separated are then placed in a vehicular fluid. This step may include, for example, the suspension in a saline solution. Block 24. It is intended that "suspension", as used herein, be interpreted broadly, with the meaning of diluted with, transported with, placed in or the like. The suspension of leukocytes in a vehicular liquid may include, for example, diluting the cells in the liquid, mixing the cells in the liquid or the like. Leukocytes may include one or more cancerous tumor cells, such as melanin.

At an optional stage of this exemplary method, several target particles are attached to the cancer cells. Block 26. The target particles or spheres may have a diameter less than 0.01 millimeters. The particles can be, for example, synthetic spheres of micrometric scale selected for their ability to be detected by the electromagnetic energy to which the sample will be exposed. By way of a particular example, gold nanoparticles, black latex spheres, stains, quantum dots or any other molecule (such as biotin) that give the cell some color that are good electromagnetic energy absorbers, such as a light, can be used To be.

The target particles may be treated to make them draw towards the cancer cell and adhere to it. Treatment may include providing surface antibodies that are known to be attracted to the analyte of interest, electrical charge or the like. In an exemplary step, the targets are joined creating a bond by an antibody-receptor pair. This provides the specificity and strength necessary for the union. The target particle is functionalized to bind to the specific antibody. These functionalized particles are introduced into the sample containing the cells with the paired receptors. After washing the remaining spheres that did not bind to a cancer cell receptor, the cancer cells have spheres attached to their receptors (usually, but not necessarily, surface receptors).

This optional stage can be useful to “amplify” the detection threshold of one or more cancer cells. An alternative step includes staining the analyte with a suitable absorbent coloration. Leftover target particles (which do not adhere to cancer cells) can be removed from the test sample by washing or other known methods. Block 28

Next, the test sample is communicated in front of a pulse laser light. Block 30. This step may include, for example, placing the sample in a system of the invention that includes a reservoir, a flow generator, such as a pump, and a conduit connecting the reservoir to a test chamber, to the The laser light is directed. When the cancer cell of interest is, for example, a melanoma, the laser light used to interrogate the fluid sample can be found anywhere in the visible range, and in other wavelengths that melanin absorbs. When the cancer cell is different from a melanoma, detection will be achieved by absorption by the target spheres.

In addition, the methods and systems of the invention may include steps and elements for using different wavelengths of light to detect particular analytes. For example, if it is known that an analyte absorbs light of the wavelength X and not Y, a test sample can be interrogated with light of a wavelength both X and Y. If a positive signal occurs when interrogated with X, but not with Y, an indication is provided that the particular analyte is present.

The laser pulse light is absorbed by melanin and induces a thermoelastic expansion in it, which results in an acoustic wave in the solution. The acoustic wave is measured using a detector such as a diaphragm. Block 32. The magnitude of the diaphragm deviation may indicate, for example, not only the presence of a cancer cell, but also an indication of its size, density, coloration, concentration and other properties. Data from the diaphragm are recorded and displayed using a controller. Block 34. This step may include, for example, amplifying an electrical signal from the diaphragm, recording it in memory and displaying it on a screen.

Having now described some general exemplary methods of the invention, a more detailed description of other methods, systems and details of embodiments of the invention can be provided. To do so, a presentation of the principles of photoacoustics in relation to some embodiments of the invention will be useful.

Photoacoustics, also called laser-induced ultrasound, use a short-lived pulsing light to create ultrasonic acoustic waves in an optically absorbent medium. These acoustic waves are generated based on the thermoelastic properties of the selected analytes as target, which in many embodiments of the invention (but not all) will comprise chromophors. Chromophores, by definition, are atoms or molecules within a compound that are responsible for the color of the given compound. Inherently, they present color by partially absorbing wavelengths that make up the visible spectrum of incident light. The wavelengths that are not absorbed are subject to dispersion and reflectance, as determined by the composition of the particular chromophore, thus emitting the representative color of the reflected wavelengths. Photoacoustic is based on the absorbed portion of the incident light. When the light is absorbed by the irradiated chromophors, the optical energy becomes kinetic thermal energy trapped inside the chromophore, and then the subsequent thermal expansion of the atoms takes place. A thermoelastic expansion occurs when a condition of confinement of tensions is achieved by depositing direct energy in a chromophore continuously, so that the energy is unable to propagate, except through the eventual convection with the surrounding media. This condition is expressed as:

t_p&lt;δ<b>/ C_s</b>

being tp the duration of the laser pulse, 8 being the absorption depth of the laser energy, and Cs being the speed of sound in the middle. It is assumed that the absorption depth, 8, is less than the diameter of the laser beam.

A transient thermoelastic expansion can be achieved by irradiating with short pulses of concentrated laser light, allowing the elastic expansion and contraction of an absorbent molecule. This thermoelastic expansion and the subsequent contraction give rise to the production of longitudinal ultrasonic waves that propagate in all directions away from the medium of thermally excited interest. A more intense heat generation will produce a more intense acoustic wave through greater thermoelastic expansion. Therefore, an intense optical absorber gives off an intense acoustic wave. Conceptually, photoacoustics can be described as pulsating laser energy that is rapidly absorbed by a dispersing medium, so that the transient thermoelastic expansion results in the formation and propagation of acoustic energy. The thermoelastic expansion, used in photoacoustics, can be described (assuming a pure absorbent in which 8 = 1 /) by

<img file="ES2708573T3_D0001.tif" />

representing p (z) the pressure at the depth z, where | Ja is the coefficient of optical absorption of the tissue, where r is the Gruneisen coefficient, which denotes the fraction of optical energy that is converted into acoustic energy. It depends on the temperature and is equal to 0.12 at room temperature for the mayone of the tissues.

The total photoacoustic energy resulting from the absorption of a beam is directly related to the chromophore content. The amount of thermoelastic expansion given in the previous equation is directly related to the absorption coefficient ja of the chromophore. The absorption coefficient is derived from the molar absorptivity coefficient and the chromophore concentration, as described with: ja = 2.3 ec, where c is the concentration of the specific chromophors and £ is the molar absorptivity coefficient of the compound. These relationships are useful to characterize the chromophore beyond its detection. For example, the magnitude of the photoacoustic energy detected may be useful for estimating the size, density and identity of an unknown chromophore.

The acoustic pressure is proportional to the energy per unit volume, given in joules per cm3. If the size of a point is kept constant, the integral of the pressure on the depth presents the total energy absorbed, given by:

<img file="ES2708573T3_D0002.tif" />

Ea is the total energy absorbed by the chromophore and Po (z) is the initial pressure as a function of the depth z. The integral gives an express amount in terms of J / cm2, so the total energy detected is related to this amount by the active area of ​​the detector. The amount of energy (Ea) absorbed is directly related to the amount of incident light energy on the medium (also given as J / cm2) up to a certain Kmite determined by the absorption capacity of the specific chromophore.

The detection of these tiny photoacoustic waves is equally as important as their production in many embodiments of the invention. Photoacoustic detection methods can vary dramatically from one experimental design to another. An element that has been found to be useful in the methods and apparatus of the invention is a piezoelectric copolymer in electrical connection with two electrodes, one being the ground wire and the other measuring the positive voltage change of the film. These films may be constructed of polyvinylidene difluoride, or PVDF, and may or may not incorporate an aluminum coating that acts as a conductive element. However, many other detector configurations are contemplated, including (by way of example and not limitation) other diaphragms whose deviation can be measured when struck by an acoustic wave, optical detectors that detect a change in the reflection index and / or of refraction in a fluid when an acoustic wave travels in it, and the like.

When the acoustic longitudinal waves propagate towards the piezoelectric film of an exemplary system and method, pressure builds up according to the magnitude of each pressure wave. When these pressure waves come into contact with the piezoelectric film, the lateral surface of the film subject to impact by the pressure wave moves, alternating the entropically stabilized bilayer. The alteration of the polymeric layer causes an electric charge to form between the two layers of the copolymer that can be detected by two conductive electrodes as a voltage peak. Using information on the magnitude of the signal (for example, the amplitude of the voltage peak) and / or the time difference between the sending of a laser pulse and the reception of the pressure wave, the density and the concentration of the chromophores they can be quantified, as well as their location relative to that of the PVDF film, as described in detail below. Some systems and methods of the invention include elements and steps of taking these measurements and using the resulting data to measure density and the like.

As discussed above, some examples of the present invention include apparatus and methods for detecting the presence of melanotic cancer cells in the human hematogenic apparatus. Some systems and methods employ photoacoustic technology as an in vitro method of detection and quantification of tumor cells in dissemination in order to detect cancers or as a method of determining the effectiveness of chemotherapy. Such devices and methods offer a way to improve the standard detection protocols, while also alleviating many of the problematic symptoms of the alternative detection methods of the prior art.

Some systems and methods of photoacoustic detection of the invention provide benefits and advantages, including the ability to accurately identify the disseminated cancer cells in the bloodstream, both early and late. This provides a relatively painless and lower cost alternative to the surgical protocols of surgical detection methods, and offers a much-needed mechanism for the early detection of the disease. It is proposed that this detection system can provide a method for precise and unprecedented detection thresholds by identifying individual melanoma cells in the presence of millions of secondary blood constituent cells.

An exemplary detection device provides a flow system through which solutions of interest can be introduced in order to excite only a single melanotic cancer cell by pulsating laser excitation. To do this, a transparent flow cell, or excitation chamber, conducive to laser excitation is incorporated into the system. Also included is a reliable acoustic detection system or a similar detection element for capturing photoacoustic waves. A signal element is used to convert the acoustic waves produced by the melanotic excitation into voltage signals that can be shown for analysis.

In FIG. 3 an exemplary detection system 50 of the invention is shown schematically. It includes, among other elements, an excitation chamber or flow cell 52. The flow cell 52 is the chamber in which laser excitation and propagation and acoustic wave detection occur. Several different flow cells are suitable for use in methods and systems of the invention. Some include transparent side walls that allow an electromagnetic energy source, the laser being an example, located externally to the cell. An exemplary cell 52 that proves to be useful is an adaptive flow cell commercially available from Spectrocell, Oreland, Pennsylvania). Cell 52 is shown schematically in the FlG. 3, and is illustrated in greater detail in FIG. Four.

FIGURES 4 (A) and 4 (B) show the flow cell 52. It has a generally rectangular three-dimensional box shape, which includes first and second opposite narrow side walls 54 joined to wider opposite side walls 56. The dimensions shown are illustrative only; other dimensions will be useful. As shown in FIG. 4B, in the exemplary cell 52 the narrow side walls have a width of approximately 2 mm (across the width of FIG. 4B) and the widest side walls have a width of approximately 10 mm. The cell 52 also includes a detection diaphragm 58 disposed on one of the widest side walls 56 and covering a cylindrical passage of approximately the same diameter in the side wall 56, whereby it is in fluid contact with the test sample inside cell 52. In exemplary cell 52, diaphragm 58 is a PVDF film with a diameter of 5 mm. The cell 52 also includes a positive electrode 60 that extends outside the interior of the cell and through a small passage in one of the narrow side walls 54 and in fluid contact with the test sample therein.

FIG. 4 (B) is a scheme showing the dimensions of the exemplary flow cell 52 and the direction of an incident laser beam 64 when it passes through cell 52 from an external source. The exemplary flow cell 52 has dimensions of approximately 2 * 10 * 45 mm (wall width 54 * wall width 56 * height) for a total fluid volume of approximately 0.9 ml. Other dimensions are contemplated and useful. Particular dimensions will be selected based on design considerations, including energy beam size, desired volumetric yield of the test sample, flow rate and the like.

The upper and lower parts of the flow chamber 52 are tapered towards cylindrical sockets 66 with an internal diameter of approximately 2.7 mm and an external diameter of approximately 4.95 mm. These sockets 66 serve to connect the cell to the conduit 68 (FIG. 3) that communicates the test sample fluid with the inside and outside of cell 52.

The high intensity laser beam 64 is directed to one of the narrow side walls 54 of the flow cell 2, opposite that of the detection diaphragm 58, at a height approximately equal to that of the detection opening 54. The laser beam 64 passes through the liquid test sample inside the cell 52 and exits through the opposite narrow side wall 54.

As discussed above, the laser beam is configured to induce a thermoelastic reaction in the test sample that results in an acoustic wave. In some methods and apparatus of the invention, the acoustic wave is detected through the use of a diaphragm 58 that deflects when the acoustic wave makes contact with it. Exemplary diaphragm 58 is in fluid contact with the test sample. The deflection of the diaphragm 58 can be detected through various methods. In the exemplary flow cell 52, the piezoelectric film diaphragm 58 is used in the wider side wall 56 of the cell as the acoustic wave sensor diaphragm.

As best illustrated in schematic of FIG. 5, the diaphragm 58 is disposed around a pair of electrodes that include the internal electrode 60 and an external electrode generally shown at 80 that will be described later. This configuration gives rise to an electric field between electrodes 60 and 80 that has to be altered and fluctuate when the diaphragm 58 is deflected between them. As will be discussed in detail hereinbelow, the fluctuation of the electric field can be measured and used to signal the presence of an acoustic wave and, therefore, of an analyte. The amount of diaphragm deviation can be used to estimate qualities of the detected analyte, including, for example, its coloration, its density, its mass and / or its size.

The piezoelectric diaphragm or film 58 in the exemplary system 50 is cut to a diameter slightly larger than a 5 mm diameter passage in one of the widest side walls 56 (10 mm) and is fixed to the outside of the flow cell 52 by a 100% silicone sealant available at DAP Inc., Baltimore, Maryland. Piezoelectric film 54 is a 100 micrometer polyvinylidene difluoride (PVDF) copolymeric film available from Ktech Corp., Albuquerque, New Mexico.

Although many additional configurations are contemplated, copper electrodes are used both for the detection of the positive signal and for the ground connection. Different mechanisms were used for each one. The exemplary positive electrode 60 is a bare copper wire 0.6 mm in diameter approximately 17 mm in length inserted into the narrow side wall 54 (2 mm wide) of the flow cell 52, as shown in FIGURES 4A- B and FIG.

5. Electrode 60 is sealed with 100% silicone sealant. It extends over a large part of the horizontal width of 10 mm of the widest side wall 56 and leaves an external end that is connected by a conductor, such as wire 82, for transfer to a controller, which may include one or more of a computer, an amplifier 84 (FIG. 3), an oscilloscope 86 (FIG. 3), or the like.

The negative electrode 80, or ground, consists of a thin circular plate 90 of copper 4 mm in diameter from Small Parts, Inc., Miami Lakes, Florida, welded to a wire of short length of 3.5 mm in diameter that , in turn, is welded to a 31-221-VP BNC 92 connector from Jameco Electronics, Belmont, California, which connects to a grounded RG 58 94 coaxial cable from Pomona Electronics, Everett, Washington. The flat copper plate 90 is flush with the outer side of the PVDF film 54. This provides a ground to the side of the piezoelectric film.

With reference once again to the scheme of FIG. 3, experimental solutions of a test sample are pumped through the circulation conduit 68 using a pump 102. In the exemplary system 50, the pump 102 is a peristaltic pump, namely a Masterflex L / S Economy Drive.

It is noted that, although many systems and methods of the invention include the circulation of test samples, others do not. Circulation is beneficial in many applications, however, since it allows a relatively large sample to be subjected to a relatively small and compact energy beam while being circulated in front of the beam.

The circulation duct 68 in the exemplary system comprises a platinum-hardened silicone tube L / S 14 available from Cole-Parmer Instruments, Vernon Hills, Illinois. Although other pumps may be used, a peristaltic pump offers advantages, because there is no mechanical element of the pump that interacts directly with the fluid sample in the conduit 100. In the exemplary system 50, approximately 15 ml of test sample solutions are introduced into an open tank 104 of 13.75 cm 3. The peristaltic pump 102 provides a negative pressure, which circulates the test sample fluid out of the reservoir 102, carrying it through from test cell 52, and finally returning it to the tank 104 through an open top. The silicone tube 68 is adjusted in size to the inlet and outlet jacks 66 (FIG. 4) of the flow cell 52. In the exemplary system 50, test sample solutions circulate at an average rate of approximately 9 ml / minute, allowing the analytes of interest to be excited 235 times when they cross vertically the beam path 64 (FIG. 4) of a laser of impulses of 5 ns with a point size of 1 mm (average conditions for a given configuration).

Those skilled in the art will appreciate that, as a source of electromagnetic energy, lasers are used, and in particular pulse laser light. There is a wide variety of lasers suitable for use in different methods and systems of the invention. For a particular application, factors such as light wavelength, beam width, intensity and the like can be specified as desired.

In the exemplary system 50, photoacoustic excitation is made possible by a carefully focused pulsed laser system shown generally at 110 in FIG. 3. Although various different lasers and other sources of electromagnetic energy can be used, the laser system 110 in the exemplary system 50 includes a neodymium doped yttrium-aluminum-garnet laser, or Nd: YAG, of triple Q switching frequency from Quantel Les Ulis , Cedex, France, housed in a vibrant integrated adjustable laser system from Opotek, Carlsbad, California. The Nd: YAG is a pumped laser that emits 1024 nm pulse laser light by means of an optical switch called "Q switch" that acts as a door to release light in the maximum inversion of neodymium ions. Once released, the laser light is reflected by two mirrors within the vibrating integrated adjustable laser system to a second harmonic generator in which the wavelength is converted to 532 nm, then to a third harmonic generator, in which it is converted into 355 nm The 355 nm laser light is pumped to an optical parametric oscillator or OPO.

The OPO contains a beta barium borate crystal that is regulated by electronically controlled rotation of the crystal with respect to the beam. The EPO converts the 355 nm input wavelength into two beams - the signal and the complementary beams - each of which has wavelengths longer than the input beam, a cavity called a double resonant oscillator oscillates the lengths of both the signal and the complementary wave, while the angle of regulation of the crystal determines a phase coincidence that produces the desired wavelength, which is then released from the cavity. The angle of regulation is controlled by a stepper motor operated by an external program generated by a computer.

The EPO allows the production of variable wavelengths ranging from 410 nm to 710 nm. The particular wavelength of the light used in the methods and systems of the invention using a laser will depend on the applications, including the wavelength that will be absorbed by the analyte being sought, and the like. As an example, a laser light of approximately 450 nm may be useful.

After the EPO, a polarizer passes through the beam. In some systems of the invention, the laser light is then coupled by a lens in a fiber optic fiber holder (an example of a standard 1.5 mm silica fiber) for light transfer. Although this may be useful, in some applications the fiber may be subject to breakage and loss of energy that results in poor results. Other exemplary devices and methods, including exemplary system 50, use an "open" delivery system, in which the laser light passes through the atmosphere from the laser system 110 to the test cell 52. In the exemplary system 50, the use of open light increased the energy input into the test cell 52, from a range of 7.0-8.0 mJ output when fiber was used, to 11-12 mJ.

In some exemplary systems and methods of the invention, there may be a compromise solution between the intensity of the radiant energy and a sufficient radiant exposure. That is, a radiant beam is, ideally, large enough to encompass all of the analyte or all chromophores that can pass through the detection cell 52, but not too large to not provide enough energy for a competent signal intensity. This compromise solution must also be balanced with the costs of a laser system 110: although it is conceivable that a system large enough to provide a very large diameter beam of great energy could be obtained, the costs for doing so may be prohibitive.

The laser beam 64 leaving the output socket of the vibrating integrated adjustable laser system 110 takes the form of an ellipse. In such exemplary methods and systems, the beam must be focused on the flow cell 52 so that the beam profile adopts a circular shape ranging from about 1 mm to 2 mm in diameter so that it enters the wall 2 mm cleanly. the flow cell 52. Different beam sizes will be useful for cells of different sizes, but it is generally advantageous to configure the beam to have a diameter that extends over the entire width of cell 52. This allows the entire cross-section of the circulating test sample be irradiated by the beam. In addition, configure the test cell in a geometry such as that shown in FIG. 4, in which the rectangular box-shaped cell has a thin side (for example, the 2 mm side) and a wider side (for example, the 10 mm side) can be advantageous. In such geometries, the beam 64 can pass through the opposite narrow side walls 54 and radiate the entire cross-section of the circulating sample within the test cell 52.

With reference again to the scheme of FIG. 3, in the exemplary system 50 the output beam 64 was spatially oriented by a series of lenses generally shown at 112. These include a cylindrical lens 114 (lens # LJ1014L2-B, of Thorlabs, Newton, New Jersey), in addition to two flat-convex collimating lenses, 100 mm (element 116) and 50 mm (element 118) of focal distance (lenses LA 1509 and LA 1131, also of Thorlabs, Newton, New Jersey) before entering flow cell 52. The result was that beam 64 has a cylindrical point size of 1 to 2 mm in diameter that mediates between 11.0-12.0 mJ when it enters cell 52. The size of the spot was determined by irradiating ZP-IT laser alignment paper, from Kentek Corp., Pittsfield, New Hampshire, with a single pulse of 5 ns and measuring the diameter of the burn.

The scheme of FIG. 6 to more fully illustrate the mechanisms of excitation and detection of some systems and methods. With regard to some exemplary methods and systems of the invention, the photoacoustic mechanism begins with the excitation of an analyte or chromophore 150; in this exemplary case, melanoma or its related tissue spectrum. In the exemplary system 50 and related exemplary methods, laser light 54 flashes with a frequency of approximately 5 ns and is at a wavelength of approximately 450 nm. The frequency of the pulses can be selected as desired, depending on factors such as geometry and cell scale, sample flow rate, analyte being searched and the like. In many exemplary systems and methods, frequencies faster than a millisecond are believed to be useful, faster than a microsecond and, in many applications (such as the exemplary system 50), of the order of one nanosecond (ns).

This bombards the chromophore 150 of interest in the test sample when it passes through the beam 64. The chromophore 150 absorbs from the beam 64 at least a portion of the incident light and the photonic energy is transformed into thermal energy. The increase in thermal energy causes a rise in temperature within chromophore 150 and a thermoelastic kinetic expansion occurs. Thermoelastic expansion is transient, due to the pulsating nature of incident laser light 64. Since the excitation pulses are short enough in duration so that no thermal heat escapes from the chromophore 150, small elastic enlargements of the cells 150 within the solution are produced, so that pressure waves 152 propagate away from the source in the range of 1-50 MHz.

Longitudinal pressure waves, such as those emanating from a photoacoustic source, propagate parallel to the direction of the wave. The resulting effect is that of a compression wave or high pressure moving band. This high pressure band moves freely in the solution (such as the environment of the test sample within the flow cell 52) away from the source in all directions. The speed of the wave varies with the application and can be, for example, approximately 5 mm per second. A portion of these waves 152 comes into contact with diaphragm 58 and hits it like a mallet hits the surface of a drum.

The PVDF film 58 is a high order copolymer that can be compared with a lipid bilayer in a biological system. In the steady state, or land, the conditions are entropically favorable for an orderly copolymeric structure. When an acoustic wave 152 hits a surface of the film 58, that copolymer layer is altered, increasing the entropy of the system and, consequently, forming a change in the altered surface. The exterior of the film 58 is grounded by the copper plate electrode 90 (FIG. 5), thereby conferring a positive charge on the inner surface of the PVDF film 58.

In other exemplary embodiments, the PVDF film or other sensor may be smaller in size, which may increase the intensity of the signal by decreasing the area of ​​the detector and eliminating part of the attenuation of the signal. Chemical products such as indium and tin oxide can be applied to the positive surface of the PVDF to increase the conductance on the surface of the film.

The positive tension produced by the photoacoustic phenomenon is conducted throughout the interior of the test cell 52 by a conductive vehicle solution, such as the 1.8% saline solution that is used in some exemplary methods and systems of the invention. The electrode 60 described above removes the charge from the flow cell 52 for further analysis.

In the exemplary system 50, the resulting electrical signal is amplified to facilitate detection. The transduced voltage of a photoacoustic effect is brought to amplifier 84 (FIG. 3), which, in the exemplary system, is a 350 MHz SR445A amplifier from Stanford Research Systems, Sunnyvale, California, in which it is amplified by four stages, each of which provides a gain of 5 for an amplification range of 5 to 125. In the exemplary system 50, the voltage signals are shown by the oscilloscope 86, which, in the exemplary system 50, is a 200 MHz TDS 2024 oscilloscope from Tektronix, Beaverton, Oregon, activated by a photodiode available at Thorlabs, Newton, New Jersey, after each laser shot.

It is noted that amplifier 84 and oscilloscope 86 are an example of electronic components useful for receiving and analyzing signals from test cell 52. In other exemplary systems, these components are replaced by a single controller, which can be, for example, a processor-based device with internal components useful for amplifying and processing the voltage signal from cell 52. Such a controller can produce data, including visual and / or audio data indicating the presence of an acoustic wave and, therefore, of a chromophore of interest. The controller can also record data in memory, and record other data, such as time, identification of the test sample and the like. The controller can be, for example, a computer.

As noted above, FIGURES 3-6 are only schematic, and have been provided to illustrate various elements of an exemplary embodiment of a system and a method of the invention. Other systems and methods will vary with respect to those illustrated above. In fact, the system, as illustrated, represents an experimental apparatus that can be configured, for example, in a laboratory. Commercial embodiments of systems and methods of the invention can be configured using alternative conduits, cells, reservoirs and the like.

By way of example, some commercial embodiments can be configured in a housing, using a computer controller that is attached to the laser, to the pump and that provides data collection, recording, processing and presentation. Some embodiments of the invention may assess portability and be configured as such. Other variations will be apparent to those skilled in the art.

As an example, an additional system of the invention was configured using an alternative configuration of the cell. The cell was configured as a dual-chamber device constructed from microscope slides available from Fisher Scientific, Pittsburgh, Pennsylvania, Loctite cyanoacrylate, Avon, Ohio, 100% Dow Coming silicone sealant, Baltimore, Maryland, and plastic sheet of nylon from Small Parts Inc., Miami Lakes, Florida. These materials were carefully cut and constructed by hand and sometimes resulted in less reliable results than those obtained with the exemplary system 50. A chamber served as a flow chamber and conducted a positive signal, similar to the flow cell 52 FIGURES 3-4. The second chamber was a stagnant tank filled with saline solution that was grounded through an electrode inserted in the chamber.

Separating the two chambers there was a glass division with a 5 mm diameter cut in it. The PVDF film is sealed on the face of the division on the grounded side. Consequently, this mechanism worked in the same way as the diaphragm in the exemplary system 50 and was used to capture the idea of ​​a proposed photoacoustic device. It was essentially consistent with cell 52 set forth above, except that the second chamber filled with saline solution replaced the grounded electrode 80.

Other variations of cell 52 have also been used. By way of example, in an alternative configuration, the internal electrode 60 was removed. This configuration used an additional positive copper plate electrode housed on the opposite side of the ground electrode 80 which held between both sides of an aluminum plated PVDF film. This film replaced the diaphragm 58 shown above. The aluminum plating can serve as conductors for both positive and ground signals, avoiding the use of the invasive electrode 60 in the flow cell.

However, it was found that the aluminum plating compromised the structural integrity of the PVDF 58 to a certain extent and resulted in a poor signal intensity. It is believed that the lack of signal intensity may have been a consequence of the short-circuit of the signal by electrode contact. To address this problem, the exemplary system 50 shown above uses the unplated film 58, an internal positive electrode 60, and a direct grounding by an external plated electrode 80 placed directly in the detection area of ​​the PVDF film 58. Although the configuration shown above was beneficial with respect to the plated film configuration, in some applications this alternate electrode configuration may be useful in some applications.

Other variations and modifications of the system and method shown above are also contemplated. Some modifications, for example, will be useful for regulating the detection threshold of the systems and methods of the invention. This can be regulated, at least to a certain extent, by adjusting the signal strength. Higher signal intensity will generally result in greater sensitivity. However, increases in signal strength must be balanced with the resulting noise in the signal. A high signal-to-noise ratio is desirable. Those skilled in the art will appreciate that the set-up of a system to achieve this will be possible and may vary in particular application details. Next, some elements of the system and of the exemplary methods that can be varied to affect the signal strength and the noise ratio are discussed.

For example, an improvement in signal intensity was achieved in the exemplary system 50 due to an increase in the size of the detection opening in the side wall 56 that underlies the diaphragm 58 from about 2 mm to about 5 mm in diameter . Generally, larger detection openings increase the likelihood of acoustic wave capture from the surrounding environment. A further improvement in signal intensity was achieved using a relatively small total cell volume, and in particular with regard to the width of the narrow side wall 54 of 2 mm. This narrow side wall width 54 of 2 mm restricts the chromophores that pass through the flow cell and forces the entire medium through the excitation beam, ensuring that no chromophore passes through the cell.

Although the value of 2 mm is only an example of a useful size, many methods and systems will benefit from using a first side wall 54 that is narrower than the second side wall 56, the source of electromagnetic energy being directed through the first narrow side wall 54. When the beam has a width that extends substantially throughout the narrow side wall 54, the entire cross section of the circulating test sample is irradiated. Directing the beam through the widest side wall 56, on the other hand, requires a significantly wider beam (with a significantly lower resulting energy per unit area).

Other configurations to achieve this result are possible, including, for example, configurations of the test cell with a narrow portion of "throat" through which the test sample flows at high speed. However, the speed should not be so great that it shortens the time of stay of the analytes in the energy beam. Next, an exemplary configuration is set forth herein.

It is also noted that there are benefits in locating the detection diaphragm 58 in the wider side wall 56, through which the beam 64 does not pass. This wider side wall 56 provides a larger area so that larger diaphragms can be used 58 In addition, in some experiments it was discovered that beam 64 could cause a greater noise in the detection signal due to a pyroelectric effect of luminous photons that interacted with the film polymers. Better performance was achieved when the beam was directed through the narrow lateral wall 54 and direct interaction with the diaphragm 58 was avoided. However, in some exemplary systems and methods, frontal excitation may prove useful.

In other embodiments of methods and systems of the invention, another modification was provided to further improve performance. For example, elements can be provided to regulate the mutual location of the laser beam 64 and cell 52. Although many possible mechanisms are possible to achieve this, a translation stage xz connected to one or both of the laser 110 or the cell is useful 52 This can be useful to ensure that the beam accurately finds test cell 52. Such a mechanism has been schematically illustrated as the XYZ adjustment device 170. In practice, this may comprise a controllable table underlying the laser 110 or cell 52 or the like.

Other potential modifications of the system 50 include the use of a fiber to communicate laser light as described above to maneuver the light source to the most efficient location for the excitation of cell 52. Test tests were performed using light emitted directly from a fiber and it was discovered that they gave rise to a point size of almost 4 mm. It was discovered that the laser light approach was important and that it was directly related to the intensity of the signal. It was also discovered that a small decrease in the diameter of the point size resulted in an increase in radiant exposure according to the inverse square law. As discussed below, it was discovered that, at least in the exemplary system 50, the use of fiber decreases the intensity of the signal.

The radiant exposure is defined as joules per cm2. With regard to the design of some systems of the invention, a decrease of one millimeter in the size of the point from 3 mm to 2 mm for a light source of 12 rnJ results in a 125% increase in radiant exposure. Therefore, a 35 mm flat convex lens (lens LAI027 of Thorlabs, Newton, New Jersey) was inserted between the fiber and the detection chamber to decrease the size of the incident point to approximately 2 mm in diameter. In the end, the entire system was repositioned for excitation without an intermediate fiber, as described above with reference to the laser system 110 and its lens 112. This increased the incident energy from 7.0 or 8.0 mJ to 11.0 or 12.0 mJ. The size of the point was taken to less than 0.026 cm3 (between 1 and 2 mm in diameter), giving a radiant exposure in the range of 460-900 mJ / cm2 and increasing the signal tensions to its highest level.

The amplification of the signal when passing from the detection apparatus to the oscilloscope can also play an important role in the identification of a signal, as well as in the noise response. In the exemplary system 50, it was found that a gain of 25 seems to produce the best signal differentiation. Often, a gain of 125 reduced the signal-to-noise ratio significantly and produced poor results, although it should be noted that, in some cases, a gain of 125 was preferable for the detection of extremely weak signals. Therefore, in the exemplary system 50, a gain of 25 is used by default; however, the gain must be fixed according to the experiment or individual application.

Detection tests were performed using the exemplary system 50 and the methods set forth above. Latex microspheres were used as a precursor to live melanoma cells. Test solutions were also used to determine a detection threshold for the exemplary system and method to characterize the sensitivity of the exemplary system. Some experimental tests were performed using a double chamber design, while the initial establishment of thresholds was carried out a unique chamber design for the flow cell.

Black CML latex microspheres (No. 2-BK-7000, available from Interfacial Dynamics Corp., Portland, Oregon) that measure 6.6 pm in diameter as tissue spectra were used to mimic the photoacoustic response of melanoma cells (the Optical properties of melanoma cells and tissue spectra are described later). The latex microspheres were opted for their broadband absorption spectrum and their relative size. A standard melanoma cell can range between 10 pm and more than 50 pm, depending on the heterogeneity of the cell line and the morphology of the cell. Although the melanoma cell has to be much larger than the microspheres used in these tests, the microspheres contain a darker pigment and, therefore, sufficiently mimic the photoacoustic effect of a melanoma cell, as discussed below. However, it is speculated that the larger size of the melanoma cells and the large number of melanin granules contained within them will allow them to produce a more intense photoacoustic signal than the microspheres per individual cell. Therefore, it is believed that the authentic melanoma will provide a signal similar to that of the microspheres, if not greater.

The spectroscopic analysis of the latex microspheres was performed using a HR-2000 high resolution spectrometer and an HL-2000 halogen light source (from Ocean Optics Inc., Dunedin, Florida). An absorption spectrum of 4.3 g / 100ml (4.3%) of black microspheres was taken using a 150 pm cuvette constructed of 1 mm thick glass slides (from Fisher Scientific, Pittsburgh, Pennsylvania) and thin material 150 pm (from Artus Corp., Englewood, New Jersey). The microsphere solution was placed in the cuvette constructed and inserted between the halogen light source and the spectrometer detector. Data were analyzed using OOIBase32 (from Ocean Optics Inc., Dunedin, Florida). Data capture was performed in an integration time of 14 ms without averaging. A short path segment was used to reduce the effects of dispersion on absorbance data. The resulting absorption spectrum confirms that the microspheres have a stable absorption throughout the visible spectrum, which demonstrates that the spheres are certainly a black broadband absorber. This spectrum indicates a slight increase in absorbance with a wavelength within the visible range.

The latex microspheres were introduced into a vehicular signal conductive fluid. In the experimental tests performed, saline solution was used. Other vehicle fluids will also be useful. Initially, the saline solution was a 0.9% sodium chloride (NaCl) solution created by adding 0.9 g of NaCl solid solute in 100 ml of deionized water solvent. In the experimental tests carried out, a 1.8% solution was used, although many other concentrations would be useful. Microspheres were added to 20 ml of saline solution in varying concentrations ranging from a maximum of 7,124 * 106 microspheres / ml to a minimum of 7.0 * 102 microspheres / ml. The microsphere concentration of each solution was deducted by:

<img file="ES2708573T3_D0003.tif" />

representing Vs the solid volume of the factory microspheres, Vm the volume of a single microsphere of 6.6 pm, Vsu the remaining volume of the factory microsphere suspension, and V the volume incorporated in the saline solution. 15 ml of each selected concentration was placed in the tank for circulation through the detection system.

Various concentrations of spheres were created using systems and methods of the invention. The tests used a 0.9% saline solution, an 510 nm excitation, an input energy of 7.5 mJ, and a dot size of 2.36 mm * 2.45 mm, resulting in a radiant exposure of 0.129 J / cm2. The signal was averaged in 64 data shots. The results of these tests confirm that the systems and methods of the invention are not only useful for detecting microspheres, but also are useful for estimating the concentration of microspheres.

Two additional sets of experiments were performed to determine the sensitivity of an exemplary system and method. In the first, concentrations of samples ranging from 7.12 * 106 to 7.0 * 102 microspheres / ml were used to obtain an understanding of the variation of the signal with the concentration. Determining this makes it possible to quantify unknown concentrations of tissue spectra by known signal intensities. This represents an important benefit of some systems and methods of the invention: signal intensity can be used to estimate qualities such as analyte density, analyte concentration, analyte size and other properties.

All tests of the first set of experiments were performed with the following experimental parameters: 450 nm excitation, 8.75 mJ energy input, 1 * 1 mm spot size, 13.1 mm beam length, radiant exposure of 0.875 J / cm2, and flow rate of 9 ml / min. Concentrations of 7.12 * 106 to 8.9 * 104 of the samples used a signal gain of 25 averaged in 64 data shots. Minor concentrations ranging from 5.5 * 103 to 7.0 * 102 used a signal gain of 125 averaged in 128 data shots. The different parameters were useful for differentiating signals from lower concentrations for this configuration. The experimental results of these experiments confirmed again that photoacoustic detection of circulating cells can be achieved using systems and methods of the invention described herein. Latex microspheres of 6.6 pm in diameter were detected successfully in 3 concentrations of the order of 106 per milliliter.

In the second set of experiments, seven microsphere solutions of different concentrations ranging from 8.9 * 104 microspheres / ml and 7.0 * 102 microspheres / ml were made to pass through a system of the invention. The tests were carried out with the following parameters: 450 nm excitation, * 25 amplification, averaged in 128 data shots, flow rate of 9 ml / min, laser energy input 11.5-12.0 mJ, point size 0.13 * 0.2 cm (0.026 cm2), and a radiant exposure of 0.461 mJ / cm2.

These experiments suggest that variations in signal amplification gain may be an exemplary parametric modification useful for increasing sensitivity. In the exemplary system 50, the gains of 25, 50 and 125 were useful, the greater gain configurations resulting in greater detection sensitivity. However, a gain setting that is too high runs the risk of a higher signal-to-noise ratio.

In additional consideration of sensitivity, assuming a homogeneous distribution of the microspheres throughout the solution, the exact amount of individual microspheres that cross the beam path at any given time can be deduced by:

<b>M </b>^,<b>= (Cm) (K </b>⁾

M # being the number of excited microspheres, Cm being the concentration of microspheres per milliliter of solution, and Vb being the volume of the path of the excitation beam through the flow cell in cm3. This deduction makes it possible to realize the number of individual cells that produce each photoacoustic signal, interpolating, therefore, the number of individual chromophors necessary to induce a differentiated signal.

In addition, during the experimental tests a more concentrated saline solution was used to enhance the intensity of the signal by increasing the conductivity by the test solution while reducing the water resistance. Initially, a 0.9% physiological saline solution had been used as the solvent for the tests. A study was carried out that doubles the saline concentration to 1.8%, with a resulting increase in signal intensity. Increasing the salt concentration from 0.9% to 1.8% increased the peak signal of the solution by a factor of 1.2. Although additional increases in intensity can be achieved with higher salt concentrations, at some level the concentration becomes too high to support the survival of a living melanoma cell, since they begin to lysate at saline concentrations above approximately 0.9%. . It is believed that concentrations of approximately 1.8% provide maximum utility, although in some circumstances higher concentrations may be used. In addition, other solutions, in addition to physiological serum, may be useful in the practice of the methods and systems of the invention. Generally, conductive fluids that support a melanoma will be useful when an acoustic sensor that relies on an internal electrode is used. Other sensor configurations may allow the use of non-conductive fluids.

Using the exemplary system set forth above with a 1.8% saline solution vehicle proved to be very successful for the photoacoustic detection of tissue spectra in the form of 6,6 pm black latex microspheres. The results indicate that the systems and methods of the invention are capable of detecting the presence of individual cells in a circulating solution.

To further characterize some systems and methods of the invention, additional tests were carried out using analytes other than the microspheres described above, or in addition to them. An application in which many systems and methods of the invention will find particular utility include (without limitation) the detection of melanoma cancer cells that circulate through the hematogenic system of a potential cancer patient. Exemplary systems and methods for detecting the invention can process materials through an in vitro analysis that requires a method to extract cells of interest from a human patient. A simple blood extraction is the most common method of obtaining cells present in the circulatory system and can be used as a routine method, relatively painless, to obtain particular samples of interest. Once a blood sample is collected, it is proposed that metastatic melanoma cells can be accurately isolated from whole blood in vitro by implementing, by way of example, a Ficoll-Hypaque centrifugation technique. This technique requires that the extracted whole blood be subjected to gradient separation by centrifugation to isolate the particular cell layer of interest before introducing it into the photoacoustic detection system.

To further evaluate the systems and methods of the invention, tissue spectra representing biological melanoma were introduced in healthy blood samples in vitro and were detected using the photoacoustic method. This section describes experimental details of such test trials. However, before providing such detail, certain background information of the application will be useful.

Leukocytes, or white blood cells, defend the body against infectious organisms and foreign agents, both in tissues and in the bloodstream itself. Human blood contains between approximately 5,000 and 10,000 leukocytes per cubic meter; the number increases in the presence of an infection. Leukocytes, like erythrocytes, are formed in bone marrow stem cells. They have nuclei and are classified into two groups: granulocytes and agranulocytes.

Granulocytes are formed in the bone marrow and represent approximately 70% of all leukocytes. Granulocytes include three types of cells: neutrophils, eosinophils and basophils. Neutrophils constitute the great mayona of granulocytes. The main purpose of these cells is to surround and destroy bacteria and other foreign particles, as well as act in inflammatory response mechanisms during infection or an allergic reaction. Granulocytes serve as the first defense line against the infection of foreign cells.

Agranulocytes include monocytes and lymphocytes. Monocytes are derived from phagocytic cells that line many vascular and lymphatic channels, called reticuloendothelial systems. Monocytes normally add up to 4% to 8% of leukocytes. They move to areas of infection, where they become macrophages, large phagocytic cells that trap and destroy organisms left by granulocytes and lymphocytes. Lymphocytes, under normal conditions, constitute between approximately 20% and 35% of all leukocytes, but they proliferate rapidly before infection. There are two basic types of lymphocytes: B lymphocytes and T lymphocytes. B lymphocytes tend to migrate to connective tissue, in which they develop in plasma cells that produce highly specific antibodies against foreign antigens. Other B lymphocytes act as memory cells, ready for a subsequent infection of the same organism. Some T lymphocytes kill invading cells directly; others interact with other cells of the immune system, regulating the immune response.

Peripheral blood mononuclear cells (PBMC) is an expression used to describe monocytes and lymphocytes that can be separated from a whole blood solution using a Ficoll-Hypaque centrifugation technique described herein. Other methods of separation, including centrifugation and similar techniques, will also be useful in the methods and systems of the invention.

Although there is conflicting information about metastatic disease and its interactions with the human immune system, it is hypothesized that the antigens present on the surface of melanoma cells in the bloodstream of an individual with metastatic disease will be recognized by these mononuclear cells , who will join them. This assumption is based on the idea that metastatic disease is a chronic disease that persists in the bloodstream and is primarily attacked by monocytes and lymphocytes, which are believed to defend against chronic disease rather than granulocytes. Therefore, isolation of the peripheral blood mononuclear cell layer should result in the isolation of any melanoma cell present in the bloodstream.

Samples of healthy and cancer-free blood were taken from individuals who gave their consent within the laboratory group. Samples were taken by venous puncture of the antecubital area of ​​the arm in an amount of 10 to 50 cubic centimeters using a standard blood collection procedure. Tubes coated with ethylenediaminetetraacetic acid (EDTA) liquid were used for blood collection to inhibit coagulation. Blood samples were stored in a refrigerated environment for no more than five hours before being processed.

A Ficoll-Hypaque separation technique was used to isolate the peripheral blood mononuclear cell layer from whole blood samples. The Ficoll-Hypaque process employs a sugar compound of a specific density that separates specific blood components by a density gradient when the centrifugal force is applied. Approximately 1 ml of Histopaque 1077 separation gradient (from Sigma-Aldrich Inc., St. Louis, Missouri) was placed in Pyrex No. 9800 glass tubes (from Corning Inc., Acton, Massachusetts). Approximately 7 ml of blood was poured from the chilled samples onto Histopaque 1077 and stopped with a rubber stopper. The sample tube was then placed in a 60 Hz Vanguard 6500 centrifuge at 3400 rpm (from Hamilton Bell Co., Montvale, New Jersey) and centrifuged for 10 minutes. The resulting gradient and relative location include the peripheral blood mononuclear cell layer, which consists of monocytes and lymphocytes separated directly above the Histopaque layer and below the plasma. The granulocytes are larger and are separated below the Histopaque layer, directly above the blood layer (not shown).

After separation, the differentiated layer of PBMC was carefully removed with standard transfer pipettes (from Samco Scientific Corp., San Fernando, California) and placed in 1.5 ml flat top microcentrifuge tubes (from Fisher Scientific , Pittsburgh, Pennsylvania). The PBMCs of the microcentrifuge tubes were washed in a saline solution and centrifuged again for 5 minutes. Leftover saline solution and plasma from the top of the PBMC layer were pipetted. This was repeated until the peripheral blood mononuclear cells were clearly isolated.

Two isolated mononuclear cell suspensions (0.111 g) were added to 20 ml of a 0.9% saline solution. A suspension served as a control. The second suspension contains 0.5 ml (1.49 * 108) of black latex microspheres. Both were introduced in an exemplary photoacoustic detection system of the invention.

A second test sample was created in which 1 ml (2.98 * 108) of 6,6 pm black latex microspheres was added to 7 ml of a human blood sample before Ficoll-Hypaque centrifugation. The peripheral blood mononuclear cell layer was isolated as previously described, 20 ml of 0.9% physiological serum was added and introduced into the detection system.

All tests were performed with the following parameters: 450 nm excitation, * 25 amplification, averaged in 128 data shots, flow rate of 9 ml / min, input energy 9.5-10.5 mJ, and a size of 0.16 * 0.16 cm (0.0256 cm2) point, resulting in a radiant exposure of 0.39 J / cm2. It is postulated that melanoma cells in the bloodstream of a metastatic patient can remain in the plasma when they are submitted to the Ficoll-Hypaque gradient. For this reason, a separate test was performed using isolated blood plasma to determine the dynamic ability of the exemplary detection system to function using different means. Two samples were prepared: a control sample consisting of 3 ml of human plasma and 10 ml of 0.9% saline solution and a test sample of the same solution with 0.5 ml (1.49 * 108) of black microspheres added, to mimic the presence of melanoma in the plasma. Experimental parameters similar to those of before were used, except that an excitation wavelength of 595 nm was used to eliminate absorption by the naturally pigmented yellow plasma. After isolation of the peripheral blood mononuclear cell layer, a physiological serum / mononuclear cell suspension was created to which 1.49 * 108 microspheres (7.12 * 106 per ml) were added. The amplification was set at 25, 450 nm excitation and photoacoustic results for a cell suspension of control agranulocytes.

The resulting output control waveform for the suspension of mononuclear cells confirms that there is no photoacoustic excitation that occurs when the PBMCs are excited. This can be expected, since the mononuclear cells are white and should not contain active chromophors that can produce a photoacoustic signal. Waveform Photoacoustic resulting from the addition of microspheres in a suspension of isolated mononuclear cells clearly confirms once again the ability of the systems and research methods investigated to identify chromophors between a circulating PBMC solution.

In addition, the waveform resulting from the addition of microspheres in whole blood prior to centrifugation is representative of the melanoma cells present in the bloodstream itself. The microspheres were added in a large concentration, so that, if all spheres were properly isolated , 7.12 * 106 microspheres / ml in the solution would result. It probably wasn't like that, since it can be assumed that many microspheres were lost during the separation process. The resulting waveform shows that Ficoll-Hypaque centrifugation is very successful in the isolation of tissue spectra that represent melanoma cells, demonstrating that this hypothesis is correct.

Blood plasma extraction illustrates a second method for detecting chromophors in a non-saline solution. The microspheres were accurately detected at high concentrations at 595 nm. This shows the versatility of the wavelength modification for detection. The 595 nm wavelength does not excite the yellow pigmented plasma (as seen in the control), mainly because the plasma absorbs in the blue and red spectrum. However, the 595 nm wavelength provides photoacoustic excitation for tissue spectra present in a solution due to the broadband absorption of the microspheres. This provides a way for the detection of isolated cells in the blood plasma, which will be beneficial.

These experiments confirmed that the photoacoustic response of tissue spectra inoculated in healthy blood can be accurately isolated and detected by the systems and methods of photoacoustic detection of the invention illustrated above. The experimental results confirm that the methods and systems of the invention are useful for extracting and identifying uniformly distributed cells among hundreds of millions of constituent blood cells that are not of interest. It also shows the ability of systems and methods to express the presence of chromophores between a suspension of mononuclear cells that eliminates the need for an additional cell isolation method. It is believed that living malignant melanoma cells will reside in the same Ficoll-Hypaque gradient as the tissue spectral cells. However, it is also possible that melanoma cells, being larger than microspheres and of different composition, can be separated differently, possibly in the granulocyte layer.

The detection assays described above have shown that the methods and systems of the invention can successfully detect chromophores in solution of the order of twenty individual cells or less. The sensitivity of an exemplary system described above is only two microspheres. Other systems and methods of the invention can be modified by selecting the laser energy, the beam area, the test cell geometry and similar parameters to achieve a detection threshold of only one cancer cell.

An additional experimental evaluation of methods and systems of the invention in applications directed to the detection of disseminated cancer cells is to test the ability to detect melanoma cells in vitro. For this, a live melanoma cell line was cultured so that a large amount of isolated melanin could be introduced into the detection system. The following exposition describes the cell line used and the cell culture methods employed, and describes in detail the methods and systems of the invention used to detect living melanoma.

Some background exposure on melanoma will be useful. Melanoma is a malignant tumor composed of unregulated melanocytes. A melanoma cell is essentially a melanocyte that contains one or more mutations that inhibit normal cell growth and regulation. A melanocyte produces coloration in mairnferos by segregating three unique pigments: dark insoluble nitrogenous eumelanins, formed by the oxidative polymerization of dihydroxyindolequinones; alkali soluble pheomelanins, derived from cysteinyl-DOPA, which provide the lightest colors (mainly brown and red-brown); and the amphoteric pheochromes (red and orange-red colors). These colors are enzymatically synthesized at 10 nm granular sites that line the inner walls of the melanocytes.

These pigments are called melanin and are produced in various forms, sizes and quantities, depending on the cell. The amount and type of melanin produced determine the color of the skin, hair and eyes in the marnfferos. The melanin produced is encapsulated by the melanoma cell that provides a pigment for any melanin producing cell. Precisely the melanin granules encapsulated within the melanocyte or melanoma cell, serve as broadband absorbers to produce photoacoustic signals when exposed to an incident laser light.

Melanins are an extensive class of functional macromolecules that jointly show a characteristic band structure model of an amorphous solid with broad band absorption spectrum in the visible and UV spectrum. It has been shown that eumelanin, the most widespread pigment, efficiently absorbs the energy of UV and visible photons and is deactivated with a quantum efficiency of less than 0.05%, which fits its role as a photoprotector in the skin. The mechanism by which this occurs is complicated and not fully understood. Studies show that melanins consist of small heterogeneous oligomeric units that have different redox states that result in a wide range of HOMO-LUMO jumps (energy difference between the highest occupied molecular orbital and the lowest unoccupied molecular orbital). This chemical disorder model allows monotone broadband absorption as a result of the superposition of a large number of extended Gaussian transitions not homogenously associated with the melanin assembly components. Despite all the work done to characterize the absorption of melanin, it has been hypothesized that the absorption spectrum of human melanin is dominated by dispersion, since it does not show characteristic absorption resonances in the visible or ultraviolet spectrum .

Melanocytes are present in all epithelial-based organs that give rise to cancerous tumors. Therefore, it is believed that melanoma cells are present in any epithelial-based cancer, or carcinomas, and their resulting metastatic cells. Unfortunately, approximately 10% of these carcinomas consist of amelanotic melanoma cells that do not produce pigment, which makes the photoacoustic detection method ineffective. However, it is believed that molecular labeling mechanisms, which include staining technology and nanoparticles, could be used as examples and not limited to identify in vitro amelanotic cells and bind to them, allowing photoacoustic excitation of the United markers. This could end up allowing the photoacoustic detection of any melanoma-based cancer.

The melanoma cell line used in the experiments set forth below was SK-MEL-1. These cells were originally obtained in 1968 from the lymph of the thoracic duct of a 29-year-old Caucasian with malignant melanoma that was advancing rapidly. Cells are known to be tumorigenic in nude mice or hamsters treated with cortisone, which produce pigmented malignant melanomas. Additional cellular features include a spherical growth property, with clusters of little niggle that presented a granular cytoplasm.

The cells were grown in suspension in 25 cm2 inclined neck flasks with a phenolic-style plug (from Corning Glass Works, Corning, New York) at 37 ° C in a humidified environment with 5% CO2. In each flask approximately 10 ml of the cell suspension was maintained. A broth consisting of 444.4 ml of RPMI (from Invitrogen Corp., Grand Island, New York), 5 ml of glutamine (also from Invitrogen Corp.), 50 ml of fetal bovine serum (from US Bio-Technologies Inc. ., Pottstown, Pennsylvania) and 0.6 ml of gentamicin (from American Pharmaceutical Partners, Schaumburg, Illinois) and was renewed three times a week. When the broth was changed, 7 ml of the cell suspension was removed and discarded, leaving 3 ml in the original flask. Then, 7 ml of fresh broth was added back to the flask.

The cells were counted before each renovation of the broth. A small sample of cells is obtained for counting. The cells were mixed gently with a blue ink with a 1: 2 solution. Using a hemocitometer, cells were counted in three squares. Cytocentrifugation was performed in a selected batch of cells and a cell block was formed and stained with hematoxylin and eosin. The individual cells varied in size, and many contained dark intracellular inclusions. The nuclei were centrally located, were round, and contained one or more prominent nucleoli.

It was found that the amount of melanin produced varied according to the cell, containing approximately 5% of the dense brown cytoplasmic granule cells uniformly dispersed throughout the cytoplasm. Therefore, only 1 in 20 live melanoma cells produce melanin actively. The mutated aneuploid state of transformed melanoma is evident through cells that contain multiple nuclei.

Several control assays were carried out using exemplary methods and systems of the invention to definitively demonstrate the detection of melanoma cells instead of other absorbents or pyroelectric effects. These control experiments were performed in addition to detecting melanoma suspensions.

In addition to a 1.8% normal saline control solution (prepared and tested according to the above exposure), a solution of 7,124 * 106 microspheres / ml was prepared using 6.6L white CML latex microspheres (from Interface Dynamics Corp., Eugene, Oregon). Although black microspheres represent the absorbing effects of melanoma, white microspheres can be used to represent the dispersing effects of melanoma cells. Therefore, the white microsphere suspension was introduced to determine the effects of a purely dispersing medium in the system. Live melanoma cells act as absorbents and dispersants due to their variable melanin content and the large amount of white surface area. It is possible that a dispersion medium (such as melanoma) may produce phantom signals or oscillations due to a pyrotechnic effect, or signals produced by scattered photons that interact with the piezoelectric film or electrodes. These control assays give examples of typical pyroelectric waveforms to eliminate the possibility of confusing a pyroelectric signal with that of a melanoma signal.

A second control experiment was performed using a 1: 1 suspension of RPMI culture broth (described above) and 1.8% saline solution. The broth, used to grow melanoma cells, contains phenol red that serves as an acid / base indicator to identify when the cells have depleted the nutrients in the culture broth. Phenol red (phenolsulfonphthalema) has a pK value of 7.9. There are different absorption spectra from different sources and detection techniques, as you can see when comparing the two spectra. It is clear that phenol red has a variable absorbance at 450 nm that could produce a photoacoustic signal, but no absorbance at 620 nm. Therefore, detection assays were performed at 450 nm and 620 nm for both suspension of the culture broth as for the melanoma suspension for comparison. This guarantees that any signal produced at 620 nm signals that of the melanoma cells, not that of the culture broth, whether present in the cells or absorbed by the cells, thus eliminating the notion that the culture medium may be producing a photoacoustic signal confused with melanoma.

Before suspending the melanoma for introduction into the detection system, the cells were counted and then centrifuged 1200 RPM at 4 ° C for ten minutes using a Fisher Scientific accuSpin 3R centrifuge. The broth supernatant was then removed from the formed cell pellet. Next, the cells were washed using Dulbeccos phosphate buffered saline solution (Invitrogen Corp., Grand Island, New York). Saline solution was added to the sediment, the cells were mixed gently, resuspended and the procedure was repeated using the same methods described above. Finally, the supernatant from the saline solution was removed and a small amount of new saline solution was added to the cell pellet.

A 15ml solution was created consisting of 2.3 * 106 live melanoma cells and 1.8% saline solution, resulting in a suspension of 1.53 * 105 cells / ml. This suspension was introduced into the exemplary system of the invention and detected using pulse laser light with the following parameters: 450 nm and 620 nm excitation, input energy of 9.5-11.6 mJ, gain of 25, and average in 128 data shots.

The cell line chosen for these assays was an ungrouped metastatic melanoma cell line. The original sample was taken from the metastatic tumor of a patient with class IV cancer, which suggests that the cells have an inherent ability to spread and enter the bloodstream. Therefore, cultured and tested cells should be comparable to cells that are present in the bloodstream of a patient with metastatic cancer. Approximately 5%, or 1 in 20, of the cultured cells produced visible melanin. The irradiated dot size for these tests was 0.13 cm * 0.2 cm (0.026 cm2) with a beam length of 1 cm. If it is assumed that 1 in 20 cells produced viable melanin, then it can be deduced that any resulting signal was produced by 200 melanoma melanoma cells per irradiated beam path. 450 nm were used to excite the melanoma samples due to the optical absorption properties presented above.

FIGURES 7A and 7B show the resulting data, including FIG. 7a the results for live melanoma and showing FIG. 7B results for the cultivation of RPMI. The noise data at the left end of each graph (near the origin) corresponds to the firing noise of the laser. As indicated in FIG. 7A, a distinct melanoma signal at approximately 2.5 ms is evident. No such peak occurs in the RPMI data of FIG. 7B. The results of these experiments confirm that the living melanoma signals clearly and systematically differed. The signals were clearly different from those of the RPMI culture broth and those of the microscopic tissue spectral tests. These characteristic waveforms clearly show the ability of the detection system to detect malignant melanoma cells in vitro.

Therefore, cultured live malignant melanoma was successfully detected using the systems and methods of the invention. The signals were clearly differentiated and retained a different pattern in relation to melanoma detection. In addition, when compared with the threshold tests for the exemplary methods and systems discussed above in relation to tests with spheres only, the melanoma signal had a tension more than 2 times greater.

The determination of the optical properties for the black latex microspheres and the living melanoma cells set forth above is useful for understanding how the tissue spectra of microspheres compare with the living cells they attempt to represent. Understanding how light interacts with the two absorbent media under test allows conclusions about its ability to produce photoacoustic signals. Once awareness of the potential for signal production is achieved through the determination of optical properties, the photoacoustic response of melanoma cell suspensions and black latex microsphere suspensions can be explained in greater detail. From these data, correlations can be established between sensitivity tests performed with latex tissue spectra and the results of the detection of real living melanoma.

Integration spheres offer a method to simultaneously determine the optical properties of materials using the inverse add-duplication algorithm to measure the combined light flux of integration spheres. This exhibition details the optical properties of interest, provides a brief explanation of the theatrical integration of the spheres measurements, describes the methodology used and exposes the optical properties of latex microspheres and malignant melanoma.

There are several properties that are used to describe the interactions of light with turbid media. The three properties considered in detail herein are absorption, dispersion and anisotropy. An important optical property for the purpose of the systems and methods of the invention is absorption. The amount of absorption of a material in a particular wavelength is directly related to the intensity of the photoacoustic signal it produces.

Absorption occurs when an incident light photon interacts with a molecule that can absorb that photon in the form of a molecular ene transition. In the visible spectrum, these transitions consist of orbital displacements of electrons in compounds formed by conjugated dienes. Compounds containing conjugated dienes constitute the mayone of the absorbent materials or chromophores. The absorption of a compound is described by its absorption coefficient, | Ja, defined as the probability of light absorption at an infinitesimal distance ds, given as cm-1. The absorption coefficient depends on the wavelength and is determined by the type of chromophore and its concentration. Assuming a purely absorbent medium, the absorption coefficient can be calculated using Beer's law, given as:

<img file="ES2708573T3_D0004.tif" />

The absorption coefficient is exponentially related to the percentage of transmitted light and depends on the thickness of the sample. This describes the simplest method of determining absorption. Realistically, there is no purely absorbent medium, which explains the need for more complicated methods of determining optical properties, such as the inverse-duplication algorithm.

The dispersion describes the interaction of photons that are not absorbed by a particular medium and is caused by changes in the refractive index in a material. The optical dispersion is described by the dispersion coefficient, Js, and is defined as the probability of photon dispersion at an infinitesimal distance ds, given by cm-1. The dispersion coefficient of a material is based on the volumetric density of the dispersers and depends on the size of the particle. Beer's law can be used in the same manner as in absorption to calculate the dispersion coefficient for a purely dispersing medium.

Anisotropy describes the amount of light that is scattered forward by a material. The phase function, or angle of refraction of light, is characterized by anisotropy, g, which is the average cosine of the phase function. Vain anisotropy between the isotropic dispersion (g = 0) and the complete frontal dispersion (g = 1).

There are other optical properties that may be relevant to some methods and systems of the invention, including, for example, dispersion albedo, given by:

<img file="ES2708573T3_D0005.tif" />

The albedo represents the relative portion of dispersion during an event of light interaction. The total light attenuation coefficient is given by:

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The effective attenuation rate for widely dispersed media is given by:

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The average free path (clm) for a photon that crosses an absorbent and dispersing medium is given by:

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The optical depth for an absorbent and dispersing medium is given by:

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Finally, the reduced coefficient of dispersion describes a very dispersive medium relating the anisotropy to the dispersion coefficient as:

<img file="ES2708573T3_D0010.tif" />

All these optical properties can be used to decipher the composition of a material through the implementation of the systems and methods of the invention.

These relationships can be used not only to better understand the operation and results of the systems and methods of the invention, but can also be used to determine and estimate the qualities of the analytes detected. The steps of a method of the invention include, for example, the use of these relationships to determine the density, mass, size of an analyte, its concentration and the like.

In addition, some other methods and systems of the invention may include steps and elements to build over time a knowledge base that is useful for determining such characteristics for analytes. A title of For example, some methods of the invention may include calibration steps, whereby different spectral targets (for example, microspheres) are tested using different concentrations, different absorbances, different input of Jan and other variations of the test parameters . A system of the invention may include a memory for storing resulting data and program instructions for analyzing the data. The resulting data can be used to develop a predictive model for use when actual test data from an unknown analyte is presented to determine the characteristics of that analyte.

In addition, so-called distinctive waveforms can be developed for different analytes by assays. These can be useful to identify an unknown analyte. In addition, some systems and methods of the invention may be useful for detecting and identifying multiple different analytes in a single sample. The different analytes present in the unique sample can be identified through their different waveforms.

The photoacoustic detection systems and methods of the invention have the ability to detect the presence of melanin in solution. The systems and methods are easy to use and allow relatively simple sample preparation, since, once a block of cells is aflapsed, only a vehicular fluid is required to be added as a saline solution to conduct the stress signal. Samples can be quickly introduced into an exemplary system through an external reservoir and circulated using a pump to induce a negative pressure. Test solutions can also be easily removed and the entire system can be cleaned in minutes, providing efficient results and high volume processing capacity.

Those skilled in the art will appreciate that many modifications can be made to the systems and methods described above. For example, detection cameras can be configured to improve the sensitivity of the device. The systems and methods of the invention have been used with spectra in the form of black CML latex microspheres of 6.6 pm, which act as a broadband absorbent similar to melanin. Photoacoustic signals derived from black latex microspheres have been discriminated against and have presented a low detection threshold, as well as a very intense and clearly differentiated signal.

The methods of the invention also include steps for the isolation of melanoma cells from whole blood. It has been discovered that the peripheral blood mononuclear cell layer can be isolated and placed in the detection system without creating false positives. In addition, the simple addition of tissue spectra in the form of latex microspheres in whole blood has produced firm results that indicate that the protocol for sample preparation can accurately isolate foreign bodies, such as melanoma, in the bloodstream. The results have proven successful in detecting broadband absorbers in the midst of millions of mononuclear cells.

An exemplary photoacoustic device of the invention has successfully detected melanoma cells in a standard saline suspension. The photoacoustic waveform for malignant melanoma is markedly different from that of other absorbents, including microspheres of tissue spectra. The acoustic diffraction of melanoma cells results in a uniquely identifiable photoacoustic waveform that can be used to differentiate a melanoma signal from possible false positives such as blood. The detection threshold is of a useful level to provide a very early detection of CTC, pathogens and other analytes of interest. In addition, modifications to some of the system elements and methods described herein are contemplated to increase sensitivity. Such modifications include, for example, increasing the intensity or size of the incident beam, decreasing the area of ​​irradiation, further improving the intensity of the signal and the like.

FIGURES 8 and 9, for example, schematically illustrate an exemplary alternative configuration of a flow cell 250. Cell 250 includes upper and lower sections 252 that are generally funnel-shaped. A narrow angle section 254 of cylindrical shape is located between the two funnel sections 252 and connects them. The angostura section is made of a transparent side wall that can be made of glass, by way of example. A laser 256 directs a beam 258 of impulses through the 256 section of angostura. As best shown in the top view of FIG. 9, the pulsating beam 258 extends substantially throughout the diameter of section 254 of cylindrical angles, whereby the entire cross section of the test sample that communicates through section 254 of angostura is illuminated. In other words, this configuration, like that of FIGURES 3-5, captures the entire flow of the test sample within the beam path.

There is an acoustic sensor 260 disposed on one side of section 254 of angostura. The sensor 260 may be a film that deflates when an acoustic wave hits it, or it may be another device. As shown in FIGURES 8 and 9, the sensor 260 is arranged in section 254 of narrowness at a location adjacent to the path of beam 258, but is located on one side where the beam does not pass directly to avoid (or at least minimize) the possible interference with or from the 258 laser beam.

The flow cell 250 can be sized as desired and as appropriate for a particular application. In an exemplary configuration, the cell is of micrometric scale, so that section 254 of angostura carries only some melanoma cells and guarantees the excitation of all the material that crosses section 254. Section 254 of angostura can be, for example, a capillary tube having a diameter of approximately 1 mm or approximately 2 mm. If provided in a suitably small diameter, it is expected that only one or some CTCs were in the small volume that resides in section 254 of Angostura. Direct the energy beam 258 to the narrow narrow 254 then excite only one or some CTC at a time.

Using a configuration such as flow cell 250 may be useful in some methods of the invention to estimate analyte concentration. Wave peaks in the resulting data can be counted to estimate the number of analyte cells detected in the sample. This knowledge, together with the volume of the sample, will lead to a determination of the analyte concentration in the sample.

The flow cell 250 can be placed in line with a reservoir, a pump and other elements, as desired. It can be used in a circulating configuration in which a sample is made to cross the section 254 of narrowness several times, or it can be used in a single-pass configuration, in which a sample crosses the flow cell 250 only one time. In an exemplary configuration, the flow cell 250 is used in a gravity flow arrangement that does not depend on a pump. Or, a syringe can be used to deliver a test sample in line with the flow cell 250, by driving the ejection pressure of the syringe the test sample through cell 250. A controller 262 is provided (FIG. 8), which is linked to both laser 256 and acoustic sensor 260. The controller can be a processor-based device, such as a computer, and includes data collection processing, data storage, laser control and acoustic sensor control functionalities. You can also include a screen to display data.

The systems and methods of detection of the invention for the detection of analytes such as metastatic melanoma have proved successful. With the ability to analyze blood samples in less than 30 minutes without the help of a trained histologist, an exemplary photoacoustic detection system can be the most reliable method of detecting cancer. This method and this unprecedented system could revolutionize the field of oncology, among others, by providing a vehicle for the early detection of a metastatic disease, in addition to functioning as a method to determine the effectiveness of chemotherapy. In addition, if the parallel teona of metastasis is true, the exemplary devices and systems will be very useful as early detectors not only of metastatic disease but also of any melanotic form of cancer near its onset.

However, the methods and systems of the invention are not limited to cancer or the detection of pathogens. Those skilled in the art will appreciate that systems and methods will find utility in a wide variety of additional applications. Some additional exemplary applications include tests to determine if a body fluid includes a particular protein or a trace of an illegal drug. Others include the detection of analytes that are present in sperm.

The benefits and advantages of the devices and systems of the invention are evident. The presentation herein of particular methods and systems has been made with the purpose of illustrating some of the best ways to implement the invention.