Analytical Performance of an Ultrasonic Particle Focusing Flow

This, in turn, greatly increases the demands on the data acquisition systems, .... the signals from all three detectors into DiDAC, a digital data acq...
0 downloads 0 Views 532KB Size
Download PDF
Anal. Chem. 2007, 79, 8740-8746

Analytical Performance of an Ultrasonic Particle Focusing Flow Cytometer Gregory R. Goddard,* Claire K. Sanders, John C. Martin, Gregory Kaduchak,† and Steven W. Graves

National Flow Cytometry Resource, Mail Stop M888, Los Alamos National Laboratory, P.O. Box 1663, Los Alamos, New Mexico 87545

Creation of inexpensive small-flow cytometers is important for applications ranging from disease diagnosis in resourcepoor areas to use in distributed sensor networks. In conventional-flow cytometers, hydrodynamics focus particles to the center of a flow stream for analysis, which requires sheath fluid that increases consumable use and waste while dramatically reducing instrument portability. Here we have evaluated, using quantitative measurements of fluorescent microspheres and cells, the performance of a flow cytometer that uses acoustic energy to focus particles to the center of a flow stream. This evaluation demonstrated measurement precision for fluorescence and side scatter CVs for alignment microspheres of 2.54% and 7.7%, respectively. Particles bearing 7 × 103 fluorophores were well resolved in a background of 50 nM free fluorophore. The lower limit of detection was determined to be about 650 fluorescein molecules. Analysis of Chinese hamster cells on the system demonstrated that acoustic focusing had no effect on cellular viability. These results indicate that the ultrasonic flow cytometer has the necessary performance for most flow cytometry applications. Furthermore, through robust engineering approaches and the combination of acoustic focusing with low-cost light sources, detectors, and data acquisition systems, it will be possible to achieve a low-cost, truly portable flow cytometer. The development of inexpensive portable medical diagnostic systems for disease diagnosis and monitoring is critical for resource poor areas of the world. This need is most exemplified for AIDS progression analysis in developing countries.1-4 As flow cytometry is the standard for AIDS progression analysis via CD4+ cell counting, there has been a large push for the introduction of low-cost portable cytometers for just this purpose.2-5 These efforts * To whom correspondence should be addressed. Phone: 505-665-0437. E-mail: [email protected]. † Present address: Acoustic Cytometry Systems, 3500 Trinity Dr., Ste. A-6, Los Alamos, NM 87544. (1) Jani, I. V.; Janossy, G.; Brown, D. W. G.; Mandy, F. Lancet Infect. Dis. 2002, 2, 243-250. (2) Janossy, G.; Jani, I. V.; Kahan, M.; Barnett, D.; Mandy, F.; Shapiro, H. Cytometry 2002, 15 (50), 78-85. (3) Mandy, F.; Bergeron, M.; Houle, G.; Bradley, J.; Fahey, J. Cytometry 2002, 15 (50), 111-116. (4) Mandy, F.; Nicholson, J.; Autran, B.; Janossy, G. Cytometry 2002, 15 (50), 39-45.

8740 Analytical Chemistry, Vol. 79, No. 22, November 15, 2007

have resulted in a number of excellent commercial offerings intended for use in resource-poor areas that include the PointCare Aurica system,6 the Guava EasyCyte,7 the Partec CyFlow,8 the Becton Dickinson FACSCount,9 the BioDETECT Microcyte,10 and just recently (though not specifically marketed as such) the Accuri C6 flow cytometer.11 In all of these cases, considerable work has gone to reducing the electronic and optical requirements of the systems through careful engineering, solid-state lasers, miniature detectors, and data acquisition systems. However, all but one of the above instruments still use the conventional flow cytometry approach wherein the sample is injected into a faster moving sheath fluid, which hydrodynamically focuses the sample into a tight stream (diameter of less than 10 µm) within the sheath stream. Typically, this approach results in the sample traveling at a high linear velocity (10 m/s) to achieve high particle analysis rates.12 This high linear velocity and the small interrogation volume (created by the intersection of the sample stream with a tightly focused laser beam) results in transit times of a few microseconds. This, in turn, greatly increases the demands on the data acquisition systems, restricts the flow channel size, requires maintenance of laminar flow for analysis, and correspondingly limits the size of the instrument.12 Nevertheless, hydrodynamic focusing positions particles with high precision to enable optimal optical interrogation. Importantly, due to the small interrogation volume, this approach also results in a largely homogeneous assay format that can resolve fluorophores in (5) Jett, J. H.; Cai, H.; Habbersett, R. C.; Keller, R. A.; Larson, E. J.; Marrone, B. L.; Nolan, J. P.; Song, X. D.; Swanson, B.; White, P. S. Flow cytometry analysis techniques for high-throughput biodefense research. In Firepower in the lab: automation in the fight against infectious diseases and bioterrorism; Layne, S. P., Beugelsdijk, T. J., Patel, C. K. N., Eds.; Joseph Henry Press: Washington, DC, 2001; pp 193-201 (Colloquium on Automation in Threat Reduction and Infectious Disease Research; April 29-30, 1999; National Academy of Science, Washington, DC). (6) Hansen, W. P. November 2005, System Overview: Aurica, www.pointcaretechnologies.net/pub/15nov05AuRICASystemOverview.pdf, 4/4/07. (7) Phi-Wilson, J.; Harvey, J.; Goix, P.; O’Neill, R. Am. Biotechnol. Lab. 2001, 19 (6), 34-36. (8) Greve, B.; Cassens, U.; Westerberg, C.; Go¨hde, jun. W.; Sibrowski, W.; Reichelt, D.; Go ¨hde, W. A Transfusion Med. Hemother. 2003, 30, 8-13. (9) Young, N. L.; Ponglertnapakorn, P.; Shaffer, N.; Srisak, K.; Chaowanachan, T.; On-Thern, V.; Kittinunvorakoon, C.; Bunwattanakul, A.; Suksaweang, S.; Pobkeeree, V.; Punnotok, J.; Mastro, T. D. Clin. Diagn Lab. Immunol. 1997, 4 (6), 783-787. (10) Kling, J Scientist 1997, 11 (13), 14. (11) Accuri Cytometers Inc., 2006; http://www.accuricytometers.com/files/ Accuri_Revolutionizes_Flow_Cytometry.pdf, 4/4/07. (12) Shapiro, H. M. Practical Flow Cytometry; John Wiley & Sons, Inc.: Hoboken, NJ, 2003. 10.1021/ac071402t CCC: $37.00

© 2007 American Chemical Society Published on Web 10/09/2007

solution versus those bound to the surface of a particle, which is typically termed resolution of free versus bound dye.13 As the use of sheath often requires liters of sheath fluid per day for operation, which increases assay cost and reduces instrument utility for field and autonomous remote operations, several attempts have been made to eliminate it. For example, early flow cytometers (Coulter counters/analyzer, the Rapid Cell spectrophotometer, etc.) did not hydrodynamically focus samples.12 This approach has been taken by one the above commercial instruments, the Guava EasyCyte, which uses a sample flowing directly through a microcapillary flow cell.7 While this approach eliminates sheath, it also greatly diminishes the system precision and its ability to perform sensitive homogeneous assays, which are the hallmarks of flow cytometry and arguably the most relevant aspects of its performance. In attempts to retain the benefits of sheath fluid but achieve miniature systems, others have attempted to miniaturize the delivery systems for hydrodynamic sheath though the use of micromechanical pumps14 or electrokinetic fluid delivery.15 These approaches of course retain the need for a storage fluid, which is detrimental to assay cost and portability. Still other approaches, such as aerodynamic focusing and recycled sheath,12,16 remove the need for stored sheath. However, these methods are also problematic. Aerodynamic focusing requires specialized hydrophilic coatings to focus the sample into a ribbon between two surfaces.16 A ribbon of sample stream is less desirable than the tight core provided by hydrodynamic focusing as it will limit the ability of the system to perform homogeneous assays. The use of recycled sheath introduces complex fluidics and additional consumables such as filters.12 Such requirements have prevented these systems from supplanting conventional hydrodynamic focusing as the standard method for sample handling in flow cytometry. In an attempt to provide a widely available viable sheathless focusing alternative to hydrodynamic focusing in a flow cytometer, we and others have developed field-focusing flow cells, using either acoustic or dielectrophoretic (DEP) fields, for use in flow cytometry.17-19 The DEP approach has shown promising results in planar geometries.19 Our acoustic approach uses ultrasonic acoustic power to tightly focus micrometer size (and above) particles to the center of a flowing stream, thereby maintaining most of the advantages of hydrodynamic focusing, while eliminating several of the shortfalls. The acoustic focusing method described in this work uses a radial ultrasonic field as the driving force to create acoustic radiation pressure (i.e., the momentum transfer from an acoustic wave to a resident particle) to move particles to the center of a flow channel. Particles within the cavity experience a time-averaged drift force that transports them to a nodal position based predominately on the contrast between the density and compress(13) Sklar, L. A.; Edwards, B. S.; Graves, S. W.; Nolan, J. P.; Prossnitz, E. R. Annu. Rev. Biophys. Biomol. Struct. 2002, 31, 97-119. (14) Sammarco, T. S.; Johnson, B. N.; Burke, D. T.; Mastrangelo, C. H.; Burns, M. A. Abstr. Pap. Am. Chem. Soc. 1996, 211 (1-2). (15) Jacobson, S. C.; and Ramsey, J. M. Anal. Chem. 1997, 69, pp. 3212-3217. (16) Huh, D.; Tung, Y. C.; Wei, H. H.; Grotberg, J. B.; Skerlos, S. J.; Kurabayashi, K.; Takayama, S. Biomed. Microdevices 2002, 4, 141-149. (17) Goddard, G.; Kaduchak, G. J. Acoust. Soc. Am. 2005, 117 (6), 3440-3447. (18) Goddard, G.; Martin, J. C.; Graves, S. W.; Kaduchak, G. Cytometry A 2006, 69 (2), 66-74. (19) Holmes, D.; Morgan, H.; Green, N. G. Biosens. Bioelectron. 2006, 21 (8), 1621.

ibility of the particles relative to the surrounding fluid.17 This cylindrically symmetric approach has been used in aerosol concentration technology20,21 and was recently adapted to liquid samples for purposes of particle concentration.17,18 Similar work has demonstrated the efficacy of acoustic concentration of particles down to submicrometer dimensions for the detection of clinically important biomolecules.22 While other acoustic approaches have been demonstrated to concentrate cells (bacteria and red blood cells) without damage,23-25 our approach has the advantage of driving the ultrasonic particle-focusing field by vibrating the entire flow chamber structure as an extended source. This type of drive methodology yields large acoustic energy transfer into the flow chamber arising through a large source aperture.17 Because the entire structure is being acoustically driven, the system performs concentration over the entire length of the acoustic flow chamber, thereby increasing the residence time of the particles in the acoustic field and allowing the use of lower power levels to drive the acoustic focusing field.17 Importantly, this acoustic approach occurs in a capillary structure that is easily adaptable into the optical train of existing flow cytometers as compared to planar structures, which will either need additional optics work or the use of epifluorescent configuration. Epifluorescent configurations are less common in flow cytometry as forward and side scatter parameters are commonly used for cellular and particle identification.12 Initial work demonstrated that the acoustic focusing approach focused particles for analysis and demonstrated that acoustic focusing might be effective in flow cytometry.18 The simplicity of its implementation along with the limited hardware requirements suggested that it would be very valuable for low-cost, portable flow cytometry instrumentation. However, the analytical performance of the initial system made it unclear whether such an approach would be suitable for most flow cytometry applications. In this work, we describe refinements to the acoustic focusing flow cell and subsequent benchmarking of the acoustic focusing flow cytometer using quantitative measurements to evaluate its performance in a flow cytometer. These measurements use commercially available biological cells and flow cytometry standard microspheres to accurately evaluate system performance, thereby establishing that the acoustic focusing flow cytometer has enough sensitivity, resolution, ability to resolve free versus bound dye, and throughput to serve as the core of a functional instrument. The implications of this approach to low-cost portable flow cytometers and the assays that could benefit from such instrumentation will also be discussed. MATERIALS AND METHODS Sample Preparation and Delivery. Reproducibility experiments were carried out using 10-µm Flow-Check microspheres from Beckman Coulter (Fullerton, CA). For determination of detection sensitivity in a fluorescent background, three bead (20) Kaduchak, G.; Sinha, D. N.; Lizon, D. C. Rev. Sci. Instrum. 2003, 73, 13321336. (21) Kaduchak, G.; Sinha, D. N. Cylindrical acoustic levitator/concentrator. U.S. Patent 6,467,350. 2002. (22) Sobanski, M. A.; Tucker, C. R.; Thomas, N. E.; Coakley, W. T. Bioseparation 2000, 9 (6), 351. (23) Yasuda, K.; Haupt, S. S.; Umemura, S. J. Acoust. Soc. Am. 1997, 102, 642645. (24) Saito, M.; Kitamura, N.; Terauchi, M. J. Appl. Phys. 2002, 92, 7581-7586. (25) Joyce, E.; Phull, S.; Lorimer, J.; Mason, T. Ultrason. Sonochem. 2003, 10 (6), 315.

Analytical Chemistry, Vol. 79, No. 22, November 15, 2007

8741

Figure 1. (A) Schematic of acoustic focusing flow cell used in acoustic flow cytometer. (B). Diagram of performance evaluation setup used for acoustic flow cytometry system

samples of 0 molecules of equivalent soluble fluorochrome26,27 (MESF), 7087 MESF, and a mixture of 0 and 7087 MESF fluorescein isothiocyanate (FITC) microspheres suspended in 50 nM FITC-Dextran. The MESF beads have a diameter of 7.8 µm and were purchased from Bangs Laboratories (Fishers, IN). All of the bead samples are suspended at a concentration of 1 × 105 particles/mL in buffered saline and vortexed to ensure homogeneous particle distribution. For cellular viability experiments, Chinese hamster cells (line CHO-K1, ATCC, Manasses, VA) were grown in R minimal essential media supplemented with 10% FBS. The CHO-K1 cells were then resuspended at a concentration of 1 × 105 cells/mL in sterile 1× PBS. All samples were driven at a typical flow rate of 0.3 mL/min using a syringe pump, Harvard Apparatus Model 22, yielding analysis rates for each sample between 100 and 200 particles/s and a measured transit time of ∼150 µs. Optics, Fluidics, and Data Acquisition. The acoustic flow cell was created as described previously.18 In short, the acoustic flow cell is the acoustic concentrator17 integrated with a square flow chamber. A top-view diagram of the flow cytometer with integrated acoustic flow cell is shown in Figure 1B. A 20-mW solidstate excitation laser (Sapphire, Coherent Inc.) operating at 488 nm is used. The laser beam is focused to a spot size of about 25 µm by 200 µm using crossed cylindrical lenses. A syringe pump, not shown in the diagram, is used to supply sample at a calibrated volumetric rate. The side scatter (SSC) and the fluorescence detector (FL1) are mounted to a optical block consisting of a microscope objective (20×, NA 0.4), adjustable aperture, two 90° removable filter blocks and a 45° removable beam splitter block. Both detectors were photomultiplier tubes (26) Gachkovskii, V. F. Dokl. Akad. Nauk SSSR 1962, 143 (1), 150-152. (27) Huynen, M. A.; Snel, B.; von Mering, C.; Bork, P.; Frishman, D.; Schwartz, A.; Wang, L.; Early, E.; Gaigalas, A.; Zhang, Y. Z.; et al. J. Res. Natl. Inst. Stand. Technol. 2002, 107 (1), 83.

8742

Analytical Chemistry, Vol. 79, No. 22, November 15, 2007

(PMTs). A small LED placed in the position of each of the PMTs is used to back illuminate the detector aperture for aligning the laser beam focus, flow chamber, and detector, by observing with a CCD camera, designated as “Alignment camera” in the diagram. A 500-nm dichroic long-pass filter (500 DCLP), optimized for 45° light incidence is used as a beam splitter. A 520-nm long-pass filter (520 LP) is used in front of the fluorescence detector (FL1) to block any 488-nm excitation laser light that is scattered from the flow chamber and particles. Similarly, a 488-nm band-pass (488/ 20 BP) filter is used in front of the side scatter detector to minimize fluorescence cross talk with the SSC detector. A ND 1.0 neutral density filter is also placed before the side scatter detector to avoid saturation of the SSC PMT and allow operation within the linear gain voltage region. A Newport model 818-UV power detector was used to capture forward scatter. An obscuration bar between the flow cell and the detector blocked the unscattered laser beam and prevented saturation of the forward scatter detector. Light scatter and fluorescence signals from each particle detected by the PMTs and photodiode are captured by triggering on the forward scatter signal from the photodiode and collecting the signals from all three detectors into DiDAC, a digital data acquisition system for flow cytometry developed within the National Flow Cytometry Resource. Data are recorded with and without the acoustic concentrating field applied. The sample fluid was cooled to ∼4 °C as a means of further increasing acoustic contrast. The rest of the experimental apparatus is equilibrated to room temperature. Acoustic power levels are ∼75 mW at 461.4 kHz. Resonance is stable throughout each experiment, but signal from a feedback transducer could be used to maintain optimal drive conditions. The acoustic concentration and analysis cuvette are diagrammed in Figure 1A. The drive and tuning transducers are both approximately 14 mm long, 1.5 mm wide, and 3 mm thick (vertical in Figure 1). The drive transducer is excited using the sine wave output from a HP 33120A function generator, which is then amplified by a Krohnhite DCA-10R power amplifier. The soft glass capillary tube is 190 mm long with inner and outer diameters of 1.9 and 3.92 mm, respectively. A glass cuvette is mechanically coupled to the end of the tube, using a pressed-fit, machined plastic coupler, and sealed using RTV silicone sealant. The glass cuvette tapers from approximately 2 mm circular i.d., 3 mm o.d. to a 2.25 mm o.d. square cuvette. The cuvette walls are 1 mm thick, yielding a 250µm square flow channel. The square cuvette provides flat optical windows for the focused laser beam and the detector optics. Data Analysis. The data are saved in FCS file format and are subsequently exported into FCS Express (De Novo Software, Thornhill, ON, Canada). Using this software, one-dimensional and bivariate histograms of peak height, width, and area were created for each sample. Coefficients of variation (CVs) were measured from these histograms. The lower limit of detection for MESF microspheres is determined as the X-intercept of the upper limit of the error bar (two standard deviations) of the detected signal from the blank microspheres.28 (28) Nguyen, D. C.; Keller, R. A.; Trkula, M. J. Opt. Soc. Am. B 1987, 4 (2). (29) Wood, J. C. S.; Hoffman, R. A. Cytometry, Part B 1998, 33 (2), 256.

Figure 2. (A) Side scatter versus fluorescence peak height with field off (B). Side scatter versus fluorescence peak area with field off (C). Side scatter versus fluorescence peak height with field on (d). Side scatter versus fluorescence peak area with field on.

RESULTS AND DISCUSSION We performed a set of experiments to benchmark the efficiency of acoustic concentration for flow cytometry on an optical platform similar to a conventional flow cytometer. Forward scatter, side scatter, and fluorescence from 10-µm Flow-Check fluorescent microspheres passing through the flow cell were captured at a sample flow rate of 0.3 mL/min. Scatter and fluorescence: peak height, width, and area measurements were collected for samples of microspheres with and without application of the acoustic focusing field. Typical data are shown in Figure 2. As can be seen in each graph in Figure 2, the data taken without the acoustic field are relatively featureless distributions, as would be expected for a homogeneous spatial distribution of particles, while data taken with the acoustic field on demonstrate a well-defined population, well-resolved from background, in both side scatter and fluorescence. Panels A and B in Figure 2 demonstrate the standard result from unfocused microspheres; as expected, there is a correlation between the intensities of side scatter and fluorescence arising from the stochastically determined particle/laser intersection point. However, as can be seen in Figure 2C and D, a typical and representative data set, peak height measurements collected with the acoustic focusing field applied demonstrated a definite population with a CV of 2.56% in fluorescence (Figure 2C), and a CV of 7.77% in side scatter. Similarly, peak area measurements collected with the focusing field applied demonstrated a definite fluorescence population with a CV of 4.53% and a side scatter population CV of 7.39% (Figure 2D). The distribution of the focused sample

population in the histogram as compared to the unfocused sample suggests that the particles were concentrated to the center from the rest of the flow cell, as expected. To verify the reproducibility of these coefficients of variation, a series of 15 samples were run with the acoustic field on. The average fluorescence CV is 3.95% with a standard deviation of the CV of 0.61%. Similarly, the average side scatter CV was 7.38% with a standard deviation of the CV of 3.23%. To ensure an accurate representation of acoustic concentration efficiency, data sets were alternately gathered in the presence and absence of the acoustic field. To evaluate the ultrasonic flow cytometer’s ability to perform homogeneous assays (resolve free vs bound dye), we mimicked a typical situation in flow cytometry where a fluorescently labeled probe is used to probe the amount of a target molecule on the surface of a particle. The volume of the particle displaces free fluorophore, and the signal is made up of the bound fluorophore as well as the autofluorescence of the polystyrene26 microsphere. In this case, the probe-target molecule has a KD in the lownanomolar range. The most common version of this experiment is an antibody binding experiment, as antibodies can be commonly obtained with these affinities for target molecules. To maintain a controlled experiment where other noninstrumental issues such as nonspecific binding could be minimized, we measured calibrated intensity FITC polystyrene microspheres bearing 0 or 7087 MESF on their surfaces in a background of 50 nM free FITC-Dextran. When the acoustic concentration field is not applied, as shown in Figure 3A, in comparison to when the Analytical Chemistry, Vol. 79, No. 22, November 15, 2007

8743

Figure 3. Detection of 7.8-µm unstained and 7087 MESF FITC microspheres in a 50 nM background of FITC. (A) Side scatter versus fluorescence peak height with field off (B) Side scatter versus fluorescence peak area with field on. (C) Histogram of fluorescence peak values with field off (D). Histogram of fluorescence peak with field on.

acoustic field is applied (Figure 3B), the two populations of FITC microspheres are easily resolved from each other, and from background in the fluorescence peak intensity contour plot. This demonstrates that we can resolve free versus bound probe at concentrations relevant to common flow cytometry experiments. With further refinement including tight optical apertures, it is expected we can work at high free dye concentrations, possibly approaching the 100-200 nM concentrations typically possible using hydrodynamically focused flow cytometers.13 To determine the limits of detection sensitivity, we collected fluorescent peak height values from Bangs Laboratories Quantum FITC MESF microsphere set containing five populations of FITC MESF microspheres at levels ranging from 0 to 44 000 molecules of FITC (Figure 4A). Figure 4B shows the background-subtracted peak height values against the MESF value of the microspheres. To achieve the highest signal-to-noise ratio, the brightest microsphere signal is off scale. Therefore, only the blank and next three brightest microsphere populations were used for the linear fit and calculations of the detection limit. Standard deviations of the peak fluorescence distribution for each particle were determined based on measurements of ∼5000 microspheres. The error bars indicate two standard deviations above and below each point for each of the microsphere sets (95% confidence level). A linear fit of the log 8744

Analytical Chemistry, Vol. 79, No. 22, November 15, 2007

data29-31 has an R2 value of 0.995. The sensitivity of the system can be estimated by using a value of two times the standard deviation of the blank bead to extrapolate the limit of detection from this fit (Figure 4B). This is similar to the approach taken by Dovichi et al.,32 and it results in an estimated lower limit of detection of 750 FITC molecules. This method inherently allows quantization of the efficacy of focusing, as a poorly focused system will have poor resolution;31 the precision of focusing must be good to achieve low detection limits else fluidic noise will dominate the measurement.33,34 Alternatively, the detection threshold method described by Wood30 is the most common method used by flow cytometer manufacturers for instrument characterization. It determines the limit of detection by extrapolating the MESF value predicted from the mean value of the blank microsphere from a linear fit of the log fluorescence values versus the log MESF values. Using this method results in a detection limit estimate of 550 MESF fluorescein molecules (Figure 4C). Both of these approaches clearly demonstrate a detection limit that compares very well to the detection limits of conventional commercial flow cytometers, which typically have sensitivities of about 300-750 fluorescein molecules.29 (30) Wood, J. C. S. Cytometry. Part B 1998, 33 (2), 260. (31) Muirhead, K. A.; Schmitt, T. C.; Muirhead, A. R. Cytometry 1983, 3, 251256.

Figure 4. Determination of the limit of detection via two approaches for the acoustic focusing flow cell flow cytometer. (A) Histogram of fluorescence peak height with field on shows 4 populations and the associated markers used to get statistical values; the fifth population is off scale. (B) Line plot of MESF value vs fluorescence (corrected for background by subtracting the mean of the blank particle) to determine the limit of detection using the 2SD method. The line is defined by the equation log y ) (log x)m + b. The dotted lines extrapolate from the fluorescence value that represents 2 standard deviations of the blank to the limit of detection level in units of MESF FITC. The bars are 2SD for each data point. (C) Line plot to determine the limit of detection using the detection threshold method. The fluorescence was uncorrected for the value of the blank. Data were fit as in (B), and the dotted lines extrapolate from the mean of the blank to the limit of detection value in MESF FITC

It should be noted that our instrument was not outfitted with extremely high numerical aperture objectives (numerical apertures of 1.3 are common on commercial systems), and our typical transit time was ∼200 µs. The relatively equal performance of our system with lower NA optics is related to the extended transit times that

acoustic focusing enables while maintaining a relatively high particle analysis rate. The use of the lower NA optics with longer working distance facilitated flexible design of the acoustic flow cell. We expect future flow cells to use higher NA optics as well as optimized data systems and detectors that will reduce electronic noise in the system, which will result in increased sensitivity and precision. Although the sample fluid is not degassed, which would be expected to raise the incidence of acoustic cavitation particularly within the frequency range used in these experiments,35-37 no cavitation is observed during the experiments. By exciting the whole cylindrical glass tube, a lower energy density within the fluid is achieved. This prevents the problem associated with cavitation in an ultrasonic field that could potentially damage fragile biological particles (e.g., red blood cells or leukocytes).22-25 Cavitation can be further alleviated by reduction of the tube diameter, as a 200-µm channel flow cell demonstrates resonance at a frequency of ∼5 MHz, well above the generally accepted 1-MHz cavitation threshold,35-37 thereby allowing even higher amplitude acoustic excitation without concern for cavitation effects. Moreover, higher frequency resonances also enable the concentration of particles to a tighter spatial distribution. To determine the effects of acoustic focusing on cellular viability, we ran unstained CHO-K1 cells with and without the acoustic field on and compared their postanalysis viability with CHO-K1 cells from the original untouched batch (Figure 5). The cells were run under the same conditions as the bead sets described above. The viability assay consisted of counting the cells from each sample, plating 200 cells from each into a 60-mm Petri dish (with 6 replicate dishes per condition). The cells were allowed to grow for 7 days; the colonies were then stained with crystal violet and counted. As can be seen in Figure 5, the cells show equal viability with and without application of the acoustic field and exhibit slightly decreased viability compared to the control cells. The slight decrease in viability is potentially due to hydrodynamic forces during the sample injection into and through the flow cell. While the present research results demonstrate the efficacy of acoustic concentration applied toward flow cytometry applications, further optimization of the experimental parameters is possible. Parameter optimization could address several aspects of the system. For this research, the optical measurements are taken using a square cuvette coupled to the acoustic concentrating flow cell with a simple but suboptimally aligned tapered transition (Figure 1B). However, the potential optimization of flow cell design, as well as a more detailed analysis of factors determining particle concentration efficiency including channel diameter, and particle analysis rate within an acoustic field can be found elsewhere.17 Furthermore, since the acoustic force on the particles is proportional to the cube of the drive frequency, smaller inner diameter acoustic focusing tubes possessing higher resonant frequencies than used for these experiments would allow for tighter focusing of the sample stream, which would improve the precision of optical measurements. Using a tighter focused laser (32) Dovichi, N. J.; Martin, J. C.; Jett, J. H.; Keller, R. A. Science 1983, 219 (4586), 845-847. (33) Graves, S. W.; Nolan, J. P.; Jett, J. H.; Martin, J. C.; Sklar, L. A. Cytometry 2002, 47 (2), 127-137. (34) Chase, E. S.; Hoffman, R. A. Cytometry. Part B 1998, 33 (2), 267.

Analytical Chemistry, Vol. 79, No. 22, November 15, 2007

8745

Figure 5. Chinese hamster cells (CHO-K1) prepared at a concentration of 1 × 105/mL, analyzed at a flow rate of 0.3 mL/min. (A) Average number of viable colonies per 200 cells after analysis for untouched and after passage through the acoustic flow cytometer with and without acoustic field concentration. (B) Forward versus side scatter peak signals for unconcentrated sample. (C) Forward versus side light scatter signals for acoustically concentrated sample.

spot would also be expected to lead to significant improvements in sensitivity and resolution. A tuning transducer, placed inline with the drive transducer to ensure resonant conditions within the fluid, could be integrated as part of a continuous feedback circuit, potentially further improving particle focusing. CONCLUSIONS The analytical performance demonstrated here clearly shows that acoustic focusing has the analytical capability to perform most flow cytometry measurements as it compares well with current commercial flow cytometry analyzers. It has this performance with the added benefits of the removal of the need for sheath, and we have previously predicted that it is possible to analyze high numbers of particles at low linear velocities due to the concentration effect of the acoustic flow cell. The low linear velocities, on the order of 10 cm/s, result in extended transit times of roughly 10-100 times slower than conventional systems, which when (35) Apfel, R. J. Acoust. Soc. Am. 1981, 69 (6), 1624-1633. (36) Flynn, H. Phys. Acoust. 1964, 1B, 57-172. (37) Neppiras, E. Acoust. Cavitation. Phys. Rep. 1980, 61, 160-251. (38) Shera, E. B.; Seitzinger, N. K.; Davis, L. M.; Keller, R. A.; Doper, S. A. Chem. Phys. Lett. 1990, 174, (6), 553-557. (39) Habbersett, R. C.; Jett, J. H. Cytometry 2004, 60A (2), 125-134. (40) Habbersett, R. C.; Naivar, M. A.; Woods, T. A.; Goddard, G. R.; Graves, S. W. Cytometry, Part A 2007, 71A, 809-817. (41) Shapiro, H. M.; Perlmutter, N. G. Cytometry 2001, 1 (44), 133-136. (42) Telford, W. G.; Hawley, T. S.; Hawley, R. G. Cytometry, Part A 2003, 4855.

8746 Analytical Chemistry, Vol. 79, No. 22, November 15, 2007

optimal optical performance is obtained should allow high particle analysis rates while providing the opportunity to collect optical signals for longer times and improve sensitivity of the measurements. Previously, extended transit time has been very significant with regards to the fluorescence detection of single molecules.38,39 Recently Habbersett et al.40 demonstrated the efficacy of using a low-cost DPSS laser and relatively inexpensive miniature PMTs. The acoustic focusing approach in combination with low-cost laser modules or other light sources such as high-power LEDS40-42 and detectors would provide most of the key components to a truly portable and low-cost flow cytometer. Such an instrument would have great impact in many areas of biomedical research and portable diagnostics. ACKNOWLEDGMENT The authors express their appreciation to Robert Habbersett and Dr. James Jett for invaluable technical discussions. We also thank Travis Woods and Dr. Michael Ward for many helpful discussions and Rebecca Hammon for technical assistance. This research was supported by NIH Grant RR020064, the National Flow Cytometry Resource (NIH Grant RR01315), and LANL Laboratory Directed Research and Development funds. Received for review July 2, 2007. Accepted August 7, 2007. AC071402T