Photoacoustic spectroscopy sample array vessels and photoacoustic spectroscopy methods for using the same

Methods and apparatus for simultaneous or sequential, rapid analysis of multiple samples by photoacoustic spectroscopy are disclosed. Particularly, a photoacoustic spectroscopy sample array vessel including a vessel body having multiple sample cells connected thereto is disclosed. At least one acoustic detector is acoustically positioned near the sample cells. Methods for analyzing the multiple samples in the sample array vessels using photoacoustic spectroscopy are provided.

FIELD OF THE INVENTION

The present invention relates to photoacoustic spectroscopic sample array vessels and photoacoustic spectroscopic analysis of samples held in such sample array vessels.

BACKGROUND

The science industry, and the bioscience and environmental industries in particular, rely on the analysis of large numbers of samples for various studies. The need for rapid turnaround time coupled with the high costs of labor and chemical waste disposal have resulted in the development of automated array-based techniques that analyze samples using optical spectroscopy. Although these conventional absorption-based techniques are applicable to a wide spectrum of analytes, they have low sensitivity.

The two major types of optical spectroscopy currently used in array-based analysis include absorption spectroscopy and fluorescence spectroscopy. The most common technique is conventional absorption spectroscopy. Light at a given wavelength is transmitted through the sample, and the decrease in intensity relative to the original beam is monitored. The concentration of absorbing substance is determined using the Beer-Lambert law, which requires knowledge of the intrinsic absorptivity of the substance, the path length of light through the sample, and the ratio of incident and transmitted light intensities. Because direct measurement of absorption involves sensing a small decrease in the strength of a high background signal (i.e., the intensity of the unblocked light beam), conventional absorption spectroscopy is a low signal-to-noise technique. As a consequence, it has limited sensitivity (typically on the order of 10−3absorbance units, corresponding to absorption of about 0.2% of the incident light by the sample). This analysis method does, however, have wide applicability. A large number of analytes absorb light with sufficient efficiency to be detected by a decrease in transmissivity.

The other major type of spectroscopy in common use with sample arrays is fluorescence spectroscopy. This technique also relies on absorption of incident light by the sample, but detection is based on the emission of light of lower energy (longer wavelength) as the absorber decays from the excited state. The background signal of the detector, therefore, is zero (except for “dark current” noise in the electronic circuitry), and the signal-to-noise is very high.

The sensitivity of fluorescence depends not only on the absorptivity of the sample, but on the intensity of the incident light and the quantum yield of the conversion of absorbed energy to fluorescent light. Under optimal conditions, fluorescent samples can be measured at a sensitivity of about 104better than conventional absorbance spectroscopy. This sensitivity, however, is achieved at the cost of versatility. Few analytes fluoresce with the yield needed for wide application of the technique. Fluorescence spectroscopy is made practical for non-fluorescing analytes by tagging them with large (e.g., ca. 500 Dalton) fluorescent molecules, thus adding an additional costly step in the overall analysis and possibly altering the chemistry of the analyte in the process.

Although both conventional absorption spectroscopy and fluorescence spectroscopy rely on the absorption of light by the analyte, they differ significantly in their sensitivity and versatility. Absorption spectroscopy is easily applied to a wide variety of analytes, but has inherently poor sensitivity. Fluorescence spectroscopy is sensitive, but only for a limited number of molecules. An array-based analysis technique is needed that combines the strengths of these two spectroscopic approaches to yield both high sensitivity and wide applicability.

Photoacoustic spectroscopy (PAS) is based on the absorption of light energy by a molecule. The signal in PAS, however, is not detected by monitoring the transmittance or emission of light. Instead, in PAS, the signal is monitored by acoustic detection. Specifically, photoacoustic spectroscopy detection is based on the generation of acoustic waves as a consequence of light absorption. Absorption of light by a sample exposed thereto excites molecules in the sample to higher rotational/vibrational/electronic states. Return to the ground state releases the absorbed energy to the surrounding medium, either as light or heat. Collisions of the molecules transfer the rotational/vibrational energy to translational energy, i.e., heat. Modulation of the light intensity (turning the light on and off as the sample is exposed) causes the temperature of the sample to rise and fall periodically. The temperature variation of the sample is accompanied by a pressure variation that creates a sound wave (gas samples must be in a closed volume). The sound wave can be detected with a sensitive microphone.

Conventionally, a sample to be analyzed by photoacoustic spectroscopy is placed in a cuvette or other similar singular sample holder. Although obtaining the advantage of PAS analysis selectivity and sensitivity, the known singular sample analysis is slow and labor intensive. There is some experimental use of PAS to analyze multiple samples, as disclosed in U.S. application Ser. No. 10/061,235, now U.S. Pat. No. 6,870,626. There is also experimental use of PAS analysis utilizing conventional microtiter plates, as disclosed in U.S. application Ser. No. 10/002,624, now U.S. Pat. No. 6,873,415, which application is incorporated herein by reference. There is still a need, however, for development of sample array vessels desired specifically for PAS analysis and for analysis parameter methods desired for PAS analysis of such sample array vessels. Further, there is a need for sample array vessels including acoustic detectors operable with PAS systems and methods that allow for PAS sample analysis using the same.

SUMMARY

Presently disclosed are PAS sample array vessels and methods for PAS sample analysis using the same. More specifically, in one embodiment a PAS sample array vessel comprises a vessel body having at least two sample cells connected to the vessel body. At least one acoustic detector capable of receiving an acoustic signal from at least one sample cell is connected or connectable to the sample array vessel. Preferably, an acoustics enhancing material is formed within spaces between the sample cells to enhance transmission of the acoustic signal from the sample cells to the acoustic detector. In another embodiment, the sample array vessel comprises a body having multiple sample cells for holding samples for PAS analysis, at least one acoustic detector positioned to detect acoustic signals that emanate from a sample in a sample cell and at least one acoustic fin acoustically connecting at least one sample cell to the acoustic detector such that the acoustic signals are directed to the detector through the acoustic fin. Various different embodiments of the sample array vessel are further described below. Also disclosed are methods for using the sample array vessels for PAS analysis.

DETAILED DESCRIPTION

Photoacoustic spectroscopy (PAS) sample array vessels and photoacoustic analysis methods are disclosed. The PAS sample array vessels and methods provide for rapid sequential or simultaneous PAS measurement of multiple samples, in various arrays, e.g., in an n, m matrix. The PAS methods and sample array vessels may be used to analyze gas, liquid, and solid samples for any type of species capable of absorbing incident electromagnetic energy. For example, the sample arrays and PAS analysis methods disclosed herein may be used to speciate various organic and inorganic transition, actinide, and lanthanide metals in solution, biomass fermentation, DNA and RNA, bacteria, to monitor serum glucose levels, and to detect pH and CO2, oil in water, water in oil, hydrogen gas, components of a gas headspace, non-destructive measurement of Cr(VI), and various species dissolved in a glass matrix.

With reference toFIG. 1, in conventional PAS apparatus2a single cuvette4holds a sample6for analysis. The sample6to be analyzed is irradiated intermittently by an excitation source8, light of a selected wavelength. The light is emitted from the excitation source8and is optically directed to a light chopper10, typically a slotted disk that rotates to effectively “switch” the light from the excitation source on and off. The intermittent light travels through optical filters12to deliver intermittent light of a selected wavelength. The light then enters the cuvette4containing the sample6. As the sample6absorbs energy from the light, acoustic waves or signals are generated. Acoustic signals are detected by an acoustic detector14. Typically, the conventional acoustic detector used is a microphone connected to the cuvette4directly or via an acoustic channel16. An amplifier18is electrically connected to the acoustic detector14to receive and amplify electrical output from the acoustic detector14.

The present PAS methods and sample arrays take advantage of the fact that the measurable physical parameter in PAS analysis is the acoustic signals generated by changes in pressure (ΔP) due to absorption of electromagnetic energy. Thus, the unique properties of PAS absorption are amenable to relatively small sample volume analyses. Specifically, the amplitude of the PAS signal, directly proportional to the ΔP, is dependent only on the density of the absorbed energy. In other words, the signal may be represented as:
Signal˜ΔP˜(β/αCpρ)(Eabs/Vo)
wherein ΔP is the pressure change in the sample, β, α, Cp, and ρ are the thermoelastic properties of the solution or matrix expansivity, compressibility, heat capacity, and density, respectively, Eabsis the quantity of energy in joules absorbed by the sample and Vois the irradiated volume (the irradiated volume is either less than or equal to the volume of the sample). As the path length is reduced both the absorbed energy and the volume are reduced simultaneously. Thus, the signal remains constant.

Furthermore, ΔP is independent of the path length. Because ΔP is a measure of the change in force per unit area, the signal is independent of the size of the detector (as long as the detector has sufficient electrical capacitance, as detailed below). Accordingly, a reduction in both the sample volume and the acoustic detector area as disclosed provides for rapid, array-based sample analyses without a loss in analytical selectivity or sensitivity. The disclosed PAS methods for simultaneous or sequential multiple sample array PAS analyses and sample array vessel apparatus also provide as much as a thousand fold increase in detection sensitivity as compared to conventional, single sample cuvettes, without sacrifice as to applicability.

More particularly, the factors affecting amplitudes of PAS signals S(V), include:1) the energy absorbed per unit excitation volume (i.e. sample volume),
Eλ(1−10−Aλ)/Vo(1)
where Eλis the energy per light pulse (J), Aλthe absorbance of the sample at the wavelength (λ) of the light pulse (dimensionless), and Vothe irradiated volume (mL);2) the thermo-elastic properties of the solution (“solvent”) containing the analyte,
β/αCpρ  (2)
where β is the thermal coefficient of expansion of the sample (° C.−1), α is the isothermal compressibility of the sample (Pa−1), Cpis the molar heat capacity of the sample (J° C.−1g−1), and ρ is the density of the sample (g mL−1);3) the acoustic geometry and transmissivity of the solvent (i.e., the solution containing an analyte of interest) and other materials used to form an acoustic bridge between the sample and a detector, e.g., a transducer,{∑i=1n⁢(ri-1/ri)⁢exp⁢[ki⁡(ri-1-ri)]⁡[1-(zi-zi+1)2/(zi+zi+1)2]}(3)
where n is the number of distinct media “i” in the acoustic bridge (including piezoelectric material in a transducer), riis the radial distance of the acoustic wave from the center of the excitation volume (m) (r0corresponds to the radius of the excitation volume), kiis the acoustic absorption coefficient for waves of known frequency and velocity in medium i (m−1), and ziis the acoustic impedance of medium “i” wherein medium i is an acoustically homogeneous medium through which sound passes en route from a source to a transducer (kg m−2s−1); and4) the efficiency of the transducer that converts the acoustic impulse into an electric impulse,
Ktwh(dtw/ε0εn)(Ct/Ccircuit)  (4)
where Ktwis the electromechanical coupling coefficient for the specific orientation of transducer (t) and acoustic wave (w), h is the thickness of the transducer element in the direction of acoustic wave travel (m), dtwis the piezoelectric strain constant for the transducer/wave orientation (C N−1), ε0is the permittivity of a vacuum (C V−1m−1), εnis the relative dielectric constant of piezoelectric material in the transducer, and Ctand Ccircuitare the respective capacitances of the transducer and the total circuit (transducer and cables) feeding into the amplifier (pF). Ktwand εnare dimensionless.

Combination of the four factors outlined above provide the following equation for expression of the signal S, assuming a cylindrical excitation (sample) volume with radial detection of a single acoustic mode in the transducer:
S=[Eλ(1−10−Aλ)/Vo][β/αCpπ]{Σni=1(ri−1/ri)exp [ki
(ri−1−ri)][1−(zi−zi+1)2/(zi+zi+1)2]}[Ktwh(dtw/ε0εn)(Ct/Ccircuit)]  (5)
PAS Sample Array Vessels

A conventional microtiter plate is shown in FIG.2. Such a conventional microtiter plate2includes a body4having multiple sample wells6. Conventional microtiter plates (such as microtiter plate2) are commercially available from sources such as MicroLiter Analytical Supplies, Millipore, and Wheaton Science Products.

FIG. 3ais a top view of an embodiment of the sample array vessel20presently disclosed (FIG. 3bis a partial side view of the same embodiment). The sample array vessel20comprises a vessel body24comprising a plate with an upper surface32and a lower surface. Vessel body24further includes multiple sample cells40formed on or connected to the plate to form an arrangement or array of sample cells40, e.g., an n, m matrix as shown inFIG. 3a.The vessel body24is formed of any sufficiently rigid material in which or to which sample cells40may be formed of or connected to, such that the sample cells are adequately supported by the vessel body. For example, the vessel body24may be formed of a plastic such as polyethylene or polypropylene.

The sample cells40may be any of a variety of shapes. For example, the sample cells may form hollow cylinders (having a bottom plate to hold samples therein) as shown inFIG. 3a.Each sample cell40comprises a well44for holding a sample to be analyzed. The sample cells40are formed of any sufficiently rigid material that is suitably transmissive to acoustic waves (e.g., a sufficiently hard material for suitable acoustic transmissibility but suitably soft so as to absorb acoustic signals generated by analytes in a sample within the sample cell40). Good results are obtained with sample array vessels20having sample cells40formed of materials, such as polystyrene, polyethylene, and/or polypropylene.

To improve acoustic transmission in the sample array vessel20from sample cell40to sample cell40, and through acoustic fins60(discussed below) to the acoustic detector, space or air gaps50(see, e.g.,FIGS. 2 and 3) between the sample cells40are preferably filled with an acoustic transmissive material. Any material capable of effectively absorbing and transmitting acoustic waves may be used. For example, good results are obtained when gaps or spaces50between the sample cells40are filled with epoxy or silicon rubber.

FIG. 14illustrates the improved acoustic transmissivity and, hence, increased acoustic signal sensitivity for sample array vessels20including acoustic gap-filling material in the gaps50between sample cells40. Specifically, a sample array vessel20having no acoustic gap-filling material has relatively low acoustic sensitivity as illustrated by curve1inFIG. 14. Asample array vessel having silicon formed in the gaps between the sample cells40shows improved acoustic transmissivity (i.e., curve2inFIG. 14) and, hence, acoustic signal sensitivity for sample cells positioned nearer the acoustic detector. A sample array vessel20having an epoxy formed in gaps between the sample cells40has improved acoustic transmissivity and, hence, acoustic signal sensitivity for all sample cells in the array vessel. The epoxy may comprise, for example, a quick-setting epoxy (available from, e.g., Radioshack as epoxy #64-231313, i.e., curve3inFIG. 14) and/or a regular setting epoxy (available from, e.g., Dexter Corp, of Olean, N.Y., as epoxy RE2038/HD3404, i.e., curve4in FIG.14). Those sample cells positioned closest to the acoustic detector (e.g., well1) have the highest level of acoustic sensitivity. A sample array vessel20having epoxy RE2038/HD3404 filling gaps between sample cells40improves acoustic transmission from all of the sample cells in the vessel and shows significantly improved acoustic transmission from sample cells positioned nearest an acoustic detector. Such a sample array vessel provides significantly increased acoustic signal sensitivity (curve4in FIG.14).

Arrangement of the sample cells40of the sample array vessel20may take any of a variety of forms. For example, a sample array vessel may have cells arranged in an n, m matrix, wherein n and m are integers from 1 to as large as can be reasonably handled by operators and PAS apparatus. Sample array vessels having about 24, 96, 384, 864, or 1536 sample cells with a 2:3 row-to-column arrangement have been found to provide good results. It is understood, however, that either n or m of the matrix may be increased to a value greater than the recited values, depending upon sample cell size, detector size, and arrangement.

The embodiment of the sample array vessel20shown inFIG. 3a,as well as the alternative embodiments discussed below, may include a single or multiple acoustic detectors48. The acoustic detectors48may comprise any suitable acoustic detector, such as microphones or transducers (e.g., piezoelectric transducers). Suitable ceramic-type transducers are available from a number of commercial sources such as PAC, Material Systems Inc., Panametrics, PCB, Stavely, or KKB. Additionally, piezoelectric crystals available from manufacturers such as Channel Industries, Sensor Technology Ltd., and Valpey Fisher or flexible polymeric transducers, such as polyvinylidenefluoride (PVDF) (commercially available from Ktech Corporation, of Albuquerque, N.Mex.) may be used. Immersion detectors (discussed below) comprising, e.g., thin probes such as VP-A50 probes available from Valpey Fisher may also be used in particular embodiments of the sample array vessels. Although acoustic detectors may be referred to at times herein as “transducers” it is to be understood that any suitable acoustic detector may be utilized.

Embodiments utilizing arrays of acoustic detectors48may require use of relatively small transducers. The transducer, however, must have sufficient capacitance, which decreases with the contact area of the transducer. Low capacitance means low ability to drive a signal through circuitry connecting the transducer to an amplifier. To maximize voltage from a transducer, contribution to Ccircuit(i.e., the associated cables and connectors of the electronic circuitry) is preferably minimized.

Of the embodiments of the sample array vessels that include one or more acoustic detectors, the acoustic detectors may be connected to or arranged relative to the sample cells40in a variety of manners. For example, to receive acoustic signals from each of the sample cells40, acoustic detectors48may be connected to or arranged relative to the sample cells40by a variety of acoustic bridges. The detectors may be positioned to receive acoustic signals from the sample cells by direct contact (e.g., contact transducers), air-coupling (e.g., air-coupled transducers) or by immersion (e.g., immersion transducersFIGS. 15a-15c).

With reference toFIG. 15a,an acoustic bridge between each sample cell140and an associated detector148may comprise a contact bridge158. The contact bridge158is formed between a wall152of the sample cell140and the detector148. An acoustic coupling fluid (e.g., grease, water, epoxy) may be placed at the contact bridge158between the detector148and an outer surface of the wall152of the sample cell140. A contact-detector arrangement preferably utilizes a transducer148in direct physical contact with the wall152of the sample cell140. Piezoelectric transducers having resonant frequencies of from about 100 KHz to about 10 MHz have been found to be especially useful.

With a direct contact design, a contact detector148(FIG. 15a), e.g., a contact transducer, preferably contacts or encapsulates at least a portion of the sample cell140. For example, the detector may comprise a hollow cylindrical transducer that fits about the outside of the wall of a sample cell40of the sample array vessel20(see, e.g., FIGS.9and10). Alternatively, the detectors may comprise flexible polymeric transducers, such as a PVDF transducers that may encompass a portion of the inside or outside wall of a sample cell or otherwise contact the sample cell or an acoustic collector post (as discussed below). Flexible detectors may accommodate slight stress differences arising from variations in sample cell geometry. A hollow-type ceramic transducer filled with a flexible, acoustically transmissive material, such as a plastic liner, may be used to minimize mechanical stress on the transducer as it fits about the outside wall of the sample cell. Although sample cells of the sample array vessel20are generally discussed herein as being cylindrical in shape, the sample cell may be of any acoustically suitable shape.

Although many embodiments of the sample array vessels are discussed and illustrated utilizing contact acoustic detectors, such as contact transducers, any suitable acoustic detector may be used with any of the embodiments of the sample array vessel. That is, any suitable acoustic bridge between the sample array vessels and acoustic detectors may be used (including but not limited to air-coupling and immersion coupling). For example, the sample array vessel20may include an air-coupling acoustic bridge162, wherein the acoustic bridge comprises air between an outer surface of a wall152of a sample cell140and an associated detector148(FIG. 15b). Detectors148that are air-coupled to the sample cell140may avoid problems associated with reproducible contact (i.e., reproducible force on the detector at the time of contact) through ensuring reproducible distance and orientation relative to the sample cell to be analyzed. Preferably, the transducer148is air-coupled near the sample cell140. An acoustic channel (104inFIG. 17) may be included to air couple the detector to the sample cell.

An illustrative embodiment of an air-coupled detection system is shown inFIG. 17, wherein a single-cell, air-coupled detection system comprises an optical window102, an acoustic detector48, and an air-column104. The detector48may be mounted to a side portion of the air column104as shown or may take any of a variety of acoustically suitable positions on the air-coupled detection system. The air-coupled detection system is positioned above a sample cell40such that a light source LB may be directed through the optical window102and into the sample cell. Any suitable sealing means, such as a non-sticking rubber gasket (not shown) may be positioned between the sample cell40of the sample array vessel and the portion of the air column104that contacts the sample array vessel surface, to ensure a suitable seal therebetween. Sealing the sample cell40to be analyzed aids in the transmission of acoustic waves from the sample cell40, through the air column104and to the detector48.

Such an air-coupled detection system may be moved from sample cell to sample cell of a sample array vessel (or the air-coupled detection system may be stationary and the sample array vessel may be moved to place the sample cells to be tested in contact with the air-coupled detection system). An air-coupled detection system (such as the embodiment shown inFIG. 17) may avoid the contamination difficulties sometimes encountered with immersion-detection systems and difficulties associated with reproducible contact sometimes encountered with contact-detection systems.

The placement of air-coupled detector embodiments relative to sample cells of a sample array vessel may be the same as or similar to that discussed below relative to contact acoustic detector placements. For example, an air-coupled detector may be fixed at each sample cell40, there may be a single or a few air-coupled detectors fixed strategically about the sample array vessel20or there may be one or more movable air-coupling detectors positionable to detect acoustic signals from sample cells40in the sample array vessel.

The sample array vessel20may alternatively have a liquid-coupling acoustic bridge, wherein an acoustic detector148is immersed in a liquid in a sample cell adjacent or near a sample cell140to be analyzed (FIG. 15c). For immersion detection, the acoustic detector preferably comprises a thin probe, such as a VP-A50 probe (available from Valpey Fisher) of a suitable size to fit within sample cell volumes of the sample array vessel20. The immersion detector may be movable such that it can be moved and placed in a sample cell immediately adjacent the sample cell to be analyzed to avoid cross contamination between samples. With such an arrangement, at least one sample cell in the sample array vessel would be sacrificed for the initial measurement of a first sample cell of the sample array vessel. Thereafter, the immersion detector would be moved and immersed in the sample cell just previously measured for analysis of the next, immediately adjacent sample cell. When cross contamination is not a concern, or when an immersion detector may be cleaned between samples, the detector may be placed directly into the cell being analyzed.

Similar to the arrangement discussed below relative to contact acoustic detectors, the sample array vessel may include an immersion detector, wherein one sample cell per row of sample cells is sacrificed and the immersion detector is positioned only in the first sample cell of the row and detects acoustic signals from all of the sample cells in the row. Alternatively, a single immersion detector may be positioned in a sample cell centrally located within the sample array vessel or about a group of sample cells within the sample array vessel. Such a sample array vessel provides a mechanically simple device as compared to a sample array vessel including the use of a movable detector.

Another type of immersion detector that may be used with the sample array vessel of the present invention may comprise an immersion transducer formed to fit within the sample cell to fit near or to contact interior walls of the sample well. For example, an array vessel having cylindrically shaped sample cells may include a hollow cylinder detector formed of a material such as PVDF or a ceramic piezoelectric that is insertable into the cylindrically shaped well. Such an arrangement avoids some impedance loss associated with a strictly aqueous acoustic contact. Further, the use of the sample fluid within the sample cell minimizes friction during transducer placement.

One or more acoustic detectors may be placed relative to the sample array vessels in any acoustically suitable position. The acoustic detectors may be attached to the sample array vessel or may be a part of a PAS system. With acoustic detectors affixed to a PAS system, the detectors may be acoustically connected to (“docked in”) the sample array vessel when the vessel is positioned within the PAS system. Whether affixed to the sample array vessel or part of a PAS system and acoustically connected to the vessel when placed in the PAS system, the acoustic detectors may be positioned in a variety of locations in or relative to the sample array vessels. The following embodiments illustrate a few of a variety of acoustic detector placements. The sample array vessels are not limited to such embodiments discussed and/or illustrated in the accompanying figures but include acoustic detectors in any of the variety of acoustically suitable detector placements.

For example, as shown inFIGS. 3aand3b,the sample array vessel20may include one or more acoustic detectors48, positioned about the perimeter of the vessel to analyze all of the sample cells40in the sample array vessel20. As shown inFIG. 3a,a first acoustic detector T1(or more) may be positioned at the end of rows m and a second acoustic detector T2(or more) may be positioned at the end of rows n (when the sample cells are arranged in a matrix). As shown, the acoustic detectors48may be connected to one or more rows of sample cells40of the sample array vessels20. The specific embodiment illustrated inFIG. 3aincludes at least two detectors T1and T2, each of which are acoustically connected to all of the sample cells40in the sample array vessel20. Thus, acoustic signals generated by analytes contained in the sample cells40would be detected by at least two detectors (i.e., T1and T2). The fewer the acoustic detectors per sample array vessel, the fewer calibration steps needed for PAS analysis of samples within the sample array vessel.

The acoustic detectors48in theFIGS. 3aand3bembodiment are preferably acoustically connected to each of the sample cells40by acoustic fins60. For the matrix sample cell arrangement shown inFIG. 3a,acoustic fins60are shown to extend both vertically and horizontally within the sample cell matrix. Acoustic fins60preferably connect each sample cell40to the next sample cell40and eventually lead to the one or more acoustic detectors48. The acoustic fins60may connect from one sample cell40to the next sample cell40by a single contact with each cell (e.g., contact with a wall of the sample cell40) or may wrap about a portion of the sample cell40wall and then lead to the next adjacent sample cell40. The acoustic fins60may extend the full height of the acoustic cell40wall.

The acoustic fins60may be formed of any sufficiently rigid material that is suitably transmissive to acoustic waves. The acoustic fin60material is preferably sufficiently hard to effectively transmit acoustic waves but suitably soft so as to absorb acoustic waves generated by analytes in a sample within the sample cell40. Acoustic fins60formed of a variety of different materials, such as polystyrene, polyethylene, polypropylene, plastics with mineral or metallic fillers (e.g., composite plastics), laminated carbon materials, metallic materials, and mixtures thereof. In alternative embodiments of the sample array vessel, the acoustic fins60perform the dual function of forming the gap50filling material discussed above as well as acting as acoustic wave paths from the sample cells to the acoustic detector(s). Gap filling material may be used in conjunction with sample array vessels having acoustic fins60to increase acoustic sensitivity of the device.

When utilizing acoustic detectors48comprising, e.g., piezoelectric transducers cylindrical in shape with a sample array vessel as shown inFIG. 3a,a substantially flat surface or end of the cylindrically-shaped transducer is (typically) the portion of the transducer that receives the acoustic signals. The transducer is, thus, preferably positioned such that the end or flat surface of the cylinder is substantially perpendicular relative to the direction in which the acoustic waves will travel. A side view of an acoustic detector that is cylindrical in shape (that may be used in theFIG. 3aembodiment) is shown inFIG. 3battached to acoustic fins60. An acoustic fin coupler61may be used to attach the acoustic fins to the acoustic detector48. Further, an acoustic coupling fluid may be placed between the acoustic fin coupler61and the acoustic detector48.

In another embodiment, as shown inFIG. 4, the sample array vessel20may include acoustic fins60forming lines along one or two sides a, b of the sample cells40in the sample array vessel. The acoustic fins60of theFIG. 4embodiment preferably extend along each row m of sample cells40from a first end52to a second end (not shown). Although the embodiment of the sample array vessel20shown inFIG. 4includes two acoustic fins60extending along either side of each row m of sample cells40, a single acoustic fin60for each row of sample cells may be used. A single acoustic fin60for each row m of sample cells40may provide a “narrower” acoustic signal to the transducer, due to possible differences in travel distances when more than one acoustic fin is utilized. Embodiments of the sample array vessel20wherein acoustic fins60are connected from one sample cell to the next (and then to the acoustic detector) in a substantially straight line (i.e., forming a limited number of (or no) turns) may reduce acoustic wave reflection and loss of acoustic signal sensitivity.

The acoustic fins60contact each sample cell at, at least, a portion of the sample cell40wall. The acoustic fins60are preferably connected to one or more acoustic detectors48positioned at the end of a set of sample cell40rows. As with all embodiments of the sample array vessel, the acoustic detectors48of the sample array vessel20embodiment shown inFIG. 4may be affixed to the sample array vessel20or may be part of the PAS system in which the sample array vessel20may be placed.

The embodiment shown inFIG. 5illustrates a directed acoustic fin60design wherein an acoustic detector48is positioned centrally in the sample array vessel or centrally about a group of sample cells40of the sample array vessel20. For example, as shown, an acoustic detector48may be centrally positioned about a group of 24 sample cells40. Thus, a sample array vessel20having 96 sample cells may include four acoustic detectors. The acoustic fins60of theFIG. 5embodiment extend from a first end62connected to a sample cell40wall to a second end63connected to the acoustic detector48. The acoustic fins60are preferably positioned such that they do not touch one another. The acoustic detectors48used may comprise, for example, a transducer embedded within the sample array vessel (or insertable into the sample array vessel). A hollow, cylindrically shaped, radially active plane-wave transducer is preferably positioned within the sample array vessel20such that the end of the cylinder is substantially parallel to (i.e., the cylinder axis is substantially perpendicular to) the plane defined by the lower surface32of the sample array vessel20.

In yet another embodiment of the sample array vessel20, as shown inFIG. 6, the vessel may include one or more detectors48, comprising, e.g., contact transducers, positioned at a collection bar64. The face of the collection bar64preferably is positioned substantially perpendicular to the lower surface32of the sample array vessel20. The collection bar64may be formed of the same material as the acoustic fins60or a different but acoustically suitable material. Each sample cell40is acoustically connected to the collection bar64(and hence the acoustic detector48) via separate acoustic fins60extending from each sample cell and terminating at the collection bar64. The acoustic fins60preferably do not contact one another. The acoustic detector48may be connected to the collection bar64, for example, using an acoustic coupling fluid and a tensioning device or support (not shown). The tension device or support maintains the acoustic detector against the collection bar64to enhance transmission of acoustic signals from the sample cells via the acoustic fins60and collection bar64to the detector. If a cylindrical acoustic detector48is used, the detector may be, for example, positioned such that an end of the cylindrical detector is substantially parallel to the face of the collection bar and perpendicular to the plane defined by the lower surface32of the sample array vessel20.

An embodiment of the collection bar64of theFIG. 6sample array vessel20is shown in FIG.7. Acoustic fins60extending from the sample cells40(seeFIG. 6) preferably terminate at the collection bar64with tapered ends66. Further, the collection bar64preferably tapers where connected to the acoustic detector48to have a cross-sectional area substantially equal to the cross-sectional area of the acoustic detector connected thereto. The tapering focuses the acoustic signals from the collection bar64to the acoustic detector48and minimizes reflection of the signals.

With reference toFIG. 8, an acoustic collector lens70may be used rather than a collection bar64(FIGS.6and7). The collector lens70focuses the acoustic signals received (via acoustic fins60) from the sample cells40to an acoustic detector48. As with the collection bar64, the acoustic collector lens70is preferably formed of the same material as the acoustic fins60of the sample array vessel20. The collector lens70may be a bulbous shape or other acoustically suitable shape that focuses acoustic waves, limits reflections, and decreases in size from a first end71to a second end72. The second end72of the collector lens70preferably has a cross-sectional area substantially equal to the cross-sectional area of the acoustic detector48to be connected thereto. The curved surface of the collector lens70allows acoustic signals to be received from the acoustic fins60attached thereto with minimal reflection and orients the acoustic waves received via the acoustic fins60toward the acoustic detector48. The acoustic signal focusing of the collector lens70embodiment increases acoustic signal sensitivity of a sample array vessel20.

Preferably, a sample array vessel20including a collector lens70also includes acoustic fins that have a first tapered end64connected to the collector lens70and a second tapered end68connected to a sample cell40. Preferably, the second tapered end68of the acoustic fins60wrap about a portion of the sample cell40to increase the amount of acoustic signal the acoustic fin60absorbs from the sample cell. Preferably, the second tapered end68of the acoustic fins60encircle about one half of the sample cell exterior to minimize acoustic signal loss through reflection. The second end68of the acoustic fins60may taper down from its initial contact point with the sample cell40to be relatively thin at about half way around the sample cell wall to further minimize back-reflection of the acoustic signal.

FIGS. 9 and 10are cross-sectional views of embodiments of the sample array vessel20. These embodiments of the vessel20include acoustic detectors48that contact each sample cell40from below. The acoustic detectors48may be moved from sample cell40to sample cell40to contact each sample cell. Alternatively, the detectors48may be affixed to a PAS system (not shown) and the sample array vessel20may be moved to connect each sample cell40with the detector sequentially.

The acoustic detector48used with the sample array vessel20ofFIG. 9may comprise, for example, a PVDF polymeric transducer. A more flexible detector minimizes problems encountered with differing cell geometries and typically has a higher sensitivity to pressure to provide more sensitivity to acoustic signal detection as compared to ceramic type detectors. In addition, the sample cells40of the sample array vessel20may be tapered as shown inFIG. 9such that the sample cells gradually increase in diameter from the lower portion of the sample cells to the upper portions of the cells.

The PVDF transducer may comprise, e.g., a ring80of PDVF material bounded by a flexible support84. The flexible support84may comprise, e.g., a slotted flexible support84bounding and supporting the acoustic detector48as shown in FIG.9. The flexible support84may, however, comprise any of a variety of mechanical devices as known to persons skilled in the art. The flexible support84ensures sufficient contact between the acoustic detector48(e.g., ring80) and the sample cell40as the detector or the sample array vessel20are moved for sequential testing of each sample cell40. As the detector48makes contact with the sample cell40the ring80slips about the outside wall of the sample cell40. Support84further ensures sufficient contact between the acoustic detector48and the wall of the sample cell40. The acoustic detector48may alternatively comprise, e.g., a ceramic cylindrical piezoelectric transducer. To protect the ceramic transducer a wear coat, e.g., a plastic liner or the like, may be placed within the detector to protect the ceramic material from forces placed upon it when slipped about different sample cells. Although theFIG. 9embodiment of the sample array vessel20shows only a single acoustic detector48, there may be multiple acoustic detectors positioned to receive a like number of sample cells so that simultaneous analysis of the sample cells40in the sample array vessel may be performed.

Another possible embodiment of a detector system is shown in FIG.10. As shown, the detector48may comprise two hemicylindrical ceramic transducers that are “clamped” about the sample cell40by a tensioning support89. (Alternatively, a pair of PVDF transducers or transducers formed of other suitable materials may be used in this embodiment.) The tensioning support89as shown inFIG. 10comprises a slotted clamping mechanism including a hinge88. The tensioning support89, when clamped or otherwise enclosed about the detector48supports the acoustic detector48and maintains contact between the detector and the sample cell. Again, the acoustic detector48is dimensioned to fit about and make contact with the outside wall of the sample cell40. As with theFIG. 9embodiment, the acoustic detector48and slotted tensioning support89may be affixed to a PAS system (not shown) and the sample array vessel20moved to analyze one or more sample cells (depending on the number of acoustic detectors present in the system). Alternatively, the acoustic detector48and slotted tensioning support89may be movable from sample cell40to sample cell40. It is to be understood that the supports84and89may comprise any of a variety of suitable support mechanisms as would be known to those persons skilled in the art and are not limited to the designs shown in FIGS.9and/or10.

As yet another alternative to the sample array vessel20embodiments shown inFIGS. 9 and 10, the acoustic detector48may instead be attached to a lower end of the flexible support84ofFIG. 9rather than comprising a ring or cylinder that fits about the sample cell40. In such an embodiment, the flexible support84(or other suitable mechanism) would fit about the sample cell40of the sample array vessel and would act to transmit the acoustic signal from the sample cell40to the acoustic detector attached to the lower end of the support84. Accordingly, the flexible support84would preferably be formed of a material similar to or the same as the material used to form the acoustic fins60of the sample array vessels discussed above. Such a device would not require the acoustic detector be of a shape and size that fits around or about the sample cell40.

With reference toFIG. 13, in yet another embodiment of the sample array vessel20, a post collector90is positioned centrally in the sample array vessel or about a group of sample cells40. Acoustic fins60lead from each sample cell40in the group of sample cells to the post collector90. Post collector90may comprise a docking station for an acoustic detector (not shown). For example, the collector post90may comprise a solid post or support that an acoustic detector contacts by, e.g., encapsulating a portion of an upper end of the collector post90(similar to the matter in which detectors may surround sample cells in the vessel shown in FIG.9). Alternatively, an acoustic detector may simply contact an end portion of the post collector90. In another embodiment, the post collector90comprises a docking station such as a hollow cylinder for receiving an acoustic detector therein. The sample array vessel20shown inFIG. 13shows only a single post collector90but may comprise multiple post collectors90positioned strategically about the array of sample cells40. The sample array vessel20ofFIG. 13can provide for sequential or simultaneous PAS analysis of groups of sample cells or of all of the sample cells in the vessel; depending upon the number of detectors included, simultaneous sampling may require that each sample cell have a different acoustic travel distance from the sample cell to the corresponding detector.

Another embodiment of the sample array vessel20is illustrated in FIG.11. The sample array vessel20includes a reflection collector bar96. The reflection collector bar96preferably forms a tapered post80extending substantially perpendicular to the lower surface32of the vessel body24, downward past the sample cells40. The reflection collector bar96may be formed as part of the body24(i.e., be integral with the vessel body) or may be connected to the body. The reflection collector bar96may include a divot98to reflect and direct acoustic waves from the sample cells40down the tapered post80of the reflection collector bar96. The divot98is preferably formed into the reflection collector bar96at an angle of about 45° C. relative to the upper surface36of the sample array vessel20.

An acoustic detector48(e.g., a piezoelectric transducer) may then be placed in contact with the reflection collector bar96and held in place by a support device, such as a spring100. Although the reflection collector bar96shown in theFIG. 11embodiment is illustrated as being positioned amongst the sample cells40, the reflection collector bar96is not limited to placement in such a location on the sample array vessel20. The reflection collector bar96may be placed on or formed by the body of the vessel in any of a variety of acoustically suitable locations; for example, the reflection collector bar96may be positioned at the end of a row of sample cells, on a comer of the sample array vessel20or there may be multiple reflection collector bars96placed in various locations on a single sample array vessel.

Another embodiment of the sample array vessel20is illustrated in FIG.12. This embodiment of the sample array vessel20is similar to that shown inFIG. 11with the exception that acoustic fins60are included to run along the side of the sample cell40walls (similar, for example, to the acoustic fins60as shown inFIGS. 5,6,8, and13) and to the reflection collector bar96.

Another embodiment of the sample array vessel includes a vessel having a sealing means, such as a cover plate to seal the sample cells of the vessel. For example, as shown inFIG. 16, a sealing plate30comprises a material transmissive to the desired incident light beam B. The sealing plate30preferably includes any suitable means31to ensure a substantially airtight seal between the sample array vessel body28and the plate30and between the plate30and an air-coupled acoustic detector48(or an acoustic-air channel as shown in FIG.17). For example, rubber gaskets or seals formed of materials such as TEFLON, KALREZ, copper, VITON, and epoxy may be used. With such a sample array vessel, as the sample is exposed to the light source, the total gas volume in the sample cell expands against the detector providing increased sensitivity. If the sealed sample array vessel were to be farther filled or compressed with a gas, e.g., helium, the acoustic sensitivity provided by the sample array vessel may be further increased.

In another embodiment of the sample array vessel20(as shown in FIG.18), the sample array vessel may simply include one to more acoustic detectors48positioned by or attached to one or more edges of the vessel body28. Alternatively, as shown inFIG. 20, an acoustic detector D may be positioned beneath the sample cell C of the sample array vessel. There may be an acoustic detector D positioned beneath each sample cell to be tested or either of the sample array vessel or the detector may be moved from one sample cell to the next. Further, the detectors may be affixed to the sample array vessel itself or may be affixed to the PAS system (not shown) upon which the sample array vessel is aligned such that one or more detectors are positioned beneath the sample cells C. In addition, the detectors D may include integrated circuitry IC to transmit the signal received by the detector to a PC (not shown).

As shown inFIG. 19, the sample array vessel20may farther include a reflective layer or plate38positioned beneath the sample cells40. The reflective layer or plate38may be connected to or connectable to the body28of the array or may simply be formed on or connected to the lower surfaces of the sample cells40. The reflective plate38may comprise any material capable of reflecting the light source or irradiation directed from a radiation source in a PAS system into samples retained within the sample cells40without significantly impeding the transfer of an acoustic signal from the sample cells40to one or more acoustic detectors48. For example, the reflective coating or plate38may comprise a metal, such as aluminum, gold, or may comprise a mirror. As shown in the embodiment illustrated inFIG. 20, a detector48may be positioned beneath each sample cell40. Further, integrated circuitry50may be attached to or connectable to each detector48. A base plate42may be included to support the integrated circuitry50, and optionally the detectors48and the reflective plate38. The sample array vessel20shown inFIG. 19may be one complete unit or may form two or more separable units. For example, the sample array vessel20may comprise unit A including the vessel body28, sample cells40and reflective plate38and unit B may comprise a base plate42including integrated circuitry50and detectors48(and, optionally, may also include reflective plate38). Unit B may be an integral part of the sample array vessel, may comprise a separable component of the array vessel or may be affixed to a PAS system whereupon the sample array vessel is aligned.

Embodiments of the sample array vessels20including acoustic detectors may further include one or more electrical interconnects (not shown) extending from the detectors48to electrically couple the detector with circuitry (not shown) for processing and/or displaying signals generated by the detector. Each detector may be electrically coupled to one or more amplifiers for amplifying signals generated by the acoustic detector.

PAS Analyses Methods

With conventional PAS sample analysis, the light source is typically modulated and monochromatized prior to reaching the sample. Modulation may be provided by mechanically chopping the light beam from a continuous source or using a pulsed source. For broad-band sources, monochromatization is achieved by use of notch filters or reflectors. For bright sources of highly absorbing samples, grating monochromators may be used. The amplitude modulation needed for Fourier-transform PAS is obtained either by scanning continuously at an optimal mirror speed or in a step-scanning mode by dithering a mirror in an interferometer during data collection.

Conventional light sources for PAS are either limited in terms of wavelength selectivity (e.g., lasers) or pulse energy (e.g., flash lamps). The pulse energy needed for adequate sensitivity for the present invention PAS methods depends upon the absorptivity of the sample to be analyzed and the efficiency of the PAS apparatus. The presently disclosed PAS methods may include use of a flash-lamp source suitable to provide a sufficient amount of energy for discrete wavelength applications. For example, the flash-lamp source may comprise a SQ xenon flash lamp available from Hamamatsu. Wavelength selectivity with minimal loss in pulse energy may be achieved with a flash-lamp source using narrow-band filters or reflectors for a limited number of wavelengths (as described in Autrey et al., Tunable UV Visible Photoacoustic Detection,Anal. Chem. Acta,(2001), which is incorporated herein by reference).

For the disclosed PAS methods, when using variable pulse photoacoustic excitation sources, preferably sufficient energy densities are deposited in the sample to yield transient pressure changes of about ≧1 mPa. Further, preferably, there is a ratio of transducer frequency (s−1) to source pulse width(s) of about ≧5. Preferably, the light source (or excitation source) has a pulse width of <5 μs and more preferably, 2 μs with pulse energy of greater than about 3 to about 30 μJ. Pulse-laser sources may also be used to practice the methods. The wave length of the light is preferably chosen based upon where the species of interest has the greatest absorbance and the sample cell material has the least absorbance, as known to those persons skilled in the art.

A notch filter may be used to provide narrow-band light. Additionally, fiber optics and reflective mirrors may be utilized to direct a light beam from the light source to a sample cell. Because the intensity of the light source decreases with increasing distance from the source, however, optical mirrors mounted on the sample array may require a standardization method (as known to those persons skilled in the art) to normalize for changes in light intensity for each sample cells in the sample array vessel. Preferably, optics to collimate the light focus on a fiber bundle, and to deliver the light beam to each sample cell in the sample array aid in the delivery of a constant path length for travel of the light beam. The light exiting such a fiber bundle is preferably shaped to provide an optimized energy density for each sample cell in the sample array vessel.

When using commercially available piezoelectric transducers for acoustic detectors, transient pressure changes in the order of 1 Pa can be detected with no electronic amplification of transducer signals. The use of low noise, high gain (e.g., 100-1000 gain) amplifiers available from Panametrics with the present methods permit the measure of mPa pressure changes with adequate signal averaging to reduce white noise. Analysis of equation (1) discussed above, showed that the photo-induced pressure change in the excitation volume (the sample volume to be analyzed) is equal to the product of the first two terms representing the density of the absorbed energy and the thermo-elastic properties of the solvent (i.e., term (1) absorbed incident light intensity per irradiated volume, term (2) expansivity, compressibility, heat capacity, and density of the solution in the sample cell). For an analyte dissolved in water, each μJ per μL of absorbed energy should result in a pressure change of about ca. 135 mPa, and thus is detectable by a piezoelectric transducer available from Airmar or Stavely. The minimum pulse energy to reach the pulse threshold of a conventional piezoelectric transducer (i.e., about 1 mPa) can be estimated when the solution absorbance and the excitation volume are known.

A light beam (or other excitation beam) is directed to the sample cell of the sample array vessel. The light beam travels into the sample in the sample cell. An acoustic wave created by absorption of energy by the sample in the sample cell is then emitted from the sample and may be detected by an acoustic detector. For example, a light path as shown inFIG. 16may be used. A light beam B is emitted from a light source (not shown) of a PAS system into a sample cell40retaining a sample. The light beam B travels into and through the sample S. If the sample array vessel includes or is being used in conjunction with a reflective plate (see, e.g., FIG.19), the light beam B is reflected back through the sample by the reflective plate. An acoustic wave created by absorption of energy by the sample is then emitted from the sample and is detected by an acoustic detector48positioned, e.g., as an air-coupled detector, positioned directly beneath the sample cell or positioned in any other of the variety of acoustically suitable locations.

Another example is illustrated inFIG. 20, using a transmissive array. A light beam B is directed from a light source (not shown) at an angle θ into and refracts through sample S (retained in a sample cell C) and through a support past an acoustic detector D. Angle θ may be less than the critical angle θc(known by those persons skilled in the art) to less than about 90° and is preferably about an angle of about 45°. An acoustic wave emitted from the irradiated sample S travels to the detector D. With such an embodiment, the light beam B is directed to avoid the detector D.

With reference toFIG. 21, in yet another example, a light beam B may be directed from a light source (not shown) in a direction substantially perpendicular to an upper surface of the sample array vessel20(or an optical reflector7may be used to direct the light beam B from a PAS system in such a direction). The light beam B is transmitted through a sample in a sample cell40. An acoustic wave AW created by absorption of energy by the sample in the sample cell40is then transmitted to the air above the sample cell and is preferably reflected by an acoustic reflector64to an acoustic detector48. In this embodiment, the acoustic reflector64transmits the excitation light beam B and reflects the acoustic waves AW.

With reference toFIG. 22, in yet another example, a light beam B is directed from a light source (not shown) in a direction substantially parallel to an upper surface of the sample array, at an angle greater than the critical angle, θc. The light beam B is reflected substantially (i.e., to produce total internal reflection “TIR”) back through a sample S (within a sample cell). The acoustic waves created by absorption of energy by the sample S is then transmitted through wall of the sample cell to an acoustic detector D. Clearly, a PAS system used with a sample array may direct the light beam to a sample in a variety of manners (in addition to those outlined herein). With the arrangements set forth inFIGS. 21 and 22, the light beams avoid impinging upon the detectors.

To analyze acoustic waveform data obtained from the transducers a statistical concept of a histogram is preferably used as described in U.S. Pat. No. 6,253,162, incorporated herein by reference.

The acoustic detectors used with the sample array vessels are preferably calibrated prior to use for detection of acoustic signals generated by analytes present within samples in the sample array vessel. A calibration method may comprise, for example, use of a standard solution or use of the acoustic detector itself in a pulse-echo mode. Specifically, a voltage is applied to the detector. The detector then expands, creating pressure within the detector and thus generates an acoustic wave that travels into the sample array vessel. The acoustic wave absorbed by and transmitted by the sample array vessel is then detected by the acoustic detector.

Alternatively, the acoustic detectors may be calibrated using a detector contact method. First, contact is made between the acoustic detector and the sample array vessel. Second, a standardized acoustic signal is generated. Third, the detector contact is adjusted until the desired electrical signal is obtained. Alternatively, the electrical signal may be detected and recorded for later analysis and normalization (as known to those persons skilled in the art). A standard acoustic signal may be generated, e.g., by directing a pulsed light onto and exposing a black or colored spot (e.g., carbon black) formed on the upper or lower surface of the sample array vessel (depending upon the sample array vessel and the position of the acoustic detector being used therewith).

A tapper calibration method may instead be used to calibrate the acoustic detectors. With the tapper method, the sample array vessel is tapped by a reproducible force to generate an acoustic wave or signal. The detector is then used to detect the signal. The sample array vessel or detector may be adjusted when calibrating using these methods (if the data is not just normalized later) by, e.g., adjusting the tension holding the detector in contact with the sample array or by other suitable means as known to those persons skilled in the art.

Whereas the invention has been described with reference to multiple embodiments of the apparatus and representative methods, it will be understood that the invention is not limited to those embodiments and representative methods. On the contrary, the invention is intended to encompass all modifications, alternatives, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.