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In-Plane Parallel Scanning: A Microarray Technology for Point-of-Care Testing Reuven Duer,*,† Russell Lund,† Richard Tanaka,† Douglas A. Christensen,‡ and James N. Herron*,‡,§ PLC Diagnostics, Inc., 192 Odebolt Drive, Thousand Oaks, California 91360, United States, Departments of Bioengineering and Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, United States A new microarray technology is described for rapid, inexpensive, multiplex diagnostics assays. Referred to as “in-plane parallel scanning” (IPPS), this technology replaces expensive laser scanning with a grid of 100-µmwide waveguides embedded in the chip’s substrate, enabling real-time quantification of molecular complex formation on the chip’s surface. Compared to conventional microarray technology, IPPS has advantages of shorter assay time and lower instrument cost and complexity so that the platform can potentially be used in point-of-care (POC) settings. Two different chip formats are described: a low-density microarray with 10 sensing wells (IPPS-10) and a medium-density one with 100 sensing wells (IPPS100). Performance was evaluated in two different proofof-principle immunoassays: interleukin-1β (IL-1β) and Clostridium difficile toxin A. The two assays gave similar limits of detection of 0.67 and 0.94 pM, respectively. A saturation kinetics model described the sensor response with apparent dissociation constants of 511 pM for IL1β and 6.47 nM for C. difficile toxin A toxoid. The multiplexing capabilities of the IPPS technology were also demonstrated in a multiplex assay for both analytes on the same IPPS-10 chip. Based on these results, the IPPS technology holds promise for translating diagnostic microarrays into near-patient environments. A recent National Institutes of Health report1 suggests that point-of-care (POC) testing has the potential to effect a paradigm shift from curative to predictive, personalized, and preemptive medicine. This shift involves the migration of key diagnostic assays from the clinical laboratory to near-patient settings, where timely diagnostic or prognostic information can help physicians make informed decisions about diagnosis and treatment options. Also, a parallel migration from clinical laboratory to the patient’s home is empowering patients to make informed decisions about * To whom correspondence should be addressed. Phone: 805-405-4620 (R.D.), (801) 581-7303 (J.N.H.). Fax: 805-265-6025 (R.D.), (801) 585-5151 (J.N.H.). E-mail:
[email protected] (R.D.),
[email protected] (J.N.H.). † PLC Diagnostics, Inc. ‡ Department of Bioengineering, University of Utah. § Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah. (1) Point-of-Care Diagnostics Testing, Fact Sheet; National Institutes of Health: Bethesda, MD, 2007; www.nih.gov/about/researchresultsforthepublic/ PointofCare.pdf. Web site access date: June 12, 2010.
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their own healthcare. These migrations started more than 20 years ago with home-use pregnancy and glucose monitoring tests, but now include more than 30 home- and professional-use tests.2,3 Biosensor technology helped enable these migrations. For example, amperometric biosensors are used in home-use glucose monitors, and electrochemical ones are employed in the professional-use POC assays used for measuring blood gases, electrolytes, glucose, hemoglobin, and metabolites.4 Additionally, optical biosensors are found in several different professional-use POC applications including cardiac biomarker assays, pulse oximetry, and transcutaneous bilirubin detection, as well as in home-use coagulation assays for self-monitoring anticoagulation therapy.4 Although these tests comprise a good first step toward a new paradigm in medical practice, none are platform technologies capable of performing different assay formats (e.g., immunoassays and molecular diagnostics assays) on a single POC instrument. In addition, many of them provide only a single diagnosis (e.g., pregnant or not), which is not sufficient for differentiating between the complex sets of biomarkers used in modern differential diagnosis of illnesses such as infectious disease or cancer. For example, the National Institute of Allergy and Infectious Disease stated in a recent report5 that “the ability to diagnose presymptomatic, symptomatic, or non-specific symptomatic individuals is essential for public health laboratories, hospital-based clinical laboratories, and point-of-care settings so that appropriate therapy can be initiated”. [Testing of pre-symptomatic individuals is probably unlikely in routine medical practice, but could have a role in triage of asymptomatic individuals following a bioterrorism event (e.g., the 2001 U.S. Capitol anthrax attack).] The same report also underscored the need for multiplex diagnostics assays that can screen a single patient sample against biomarkers specific for different pathogens, as well as multiple, independent biomarkers for the same pathogen. (2) Henley, L. Point-of-Care Testing; MedSun Newsletter 26; Food and Drug Administration: Silver Spring, MD, 2008; http://www.accessdata.fda.gov/ scripts/cdrh/cfdocs/medsun/news/printer.cfm?id)817. Web site access date: June 12, 2010. (3) Warsinke, A. Anal. Bioanal. Chem. 2009, 393, 1393–405. (4) Herron, J. N.; Wang, H.-K.; Tan, L.; Brown, S. Z.; Terry, A. H.; Durtschi, J. D.; Tolley, S. E.; Simon, E. M.; Astill, M. E.; Smith, R. S.; Christensen, D. A. In Fluorescence Sensors and Biosensors; Thompson, R. B., Ed.; Taylor & Francis/CRC Press: Boca Raton, FL, 2005; pp 283-332. (5) Advanced Product Development for Multiplex Infectious Disease Diagnostics: Summary of a NIAID Workshop held on June 27, 2005; National Institute of Allergy and Infectious Disease: Bethesda, MD, 2006; http://www.niaid.nih. gov/topics/diagnostics/Documents/adv_prod.pdf. Web site access date: October 11, 2010. 10.1021/ac101571b 2010 American Chemical Society Published on Web 10/14/2010
Similar challenges are faced in developing multiplex POC tests for early-stage cancer detection. For example, an immunoproteomics approach is being developed for early-stage ovarian cancer that uses panels of 50 or more tumor-associated antigens to detect circulating autoantibodies that bind these antigens.6,7 The immune system produces such antibodies early in oncogenesis when patients are still asymptomatic. There is a compelling medical need for such tests because CA125, the most commonly measured biomarker for ovarian cancer, has little predictive value for earlystage disease.6,8 However, the immunoproteomics work is relative early stage, involving identification of clinically relevant tumorassociated antigens and demonstration of their clinical sensitivity and selectivity. A recent advancement in this area illustrates how biosensor technology can facilitate migration of early-stage cancer detection into near-patient settings. In particular, Salama et al.9 reported a single-analyte chemiluminescence assay for autoantibodies to a breast and ovarian cancer-associated antigen (GIPC1) based on optical-fiber biosensor technology. This biosensor had a significantly lower detection limit for an anti-GIPC-1 autoantibody than comparable chemiluminescent enzyme-linked immunosorbant assay (ELISA) technology (30 pg/mL for biosensor versus 1.5 ng/mL for ELISA). It also exhibited significantly higher clinical sensitivity (77% for biosensor versus 27% for ELISA) in a smallscale (22-patient) clinical study. Moreover, it has multiplex assay capabilities and can be deployed as a handheld analyzer in POC settings. Another multiplex diagnostics approach is based on the correlation between gene expression signatures and the activation of oncogenic signaling pathways.10 Gene expression panels of up to 70 oncogenic signaling pathways are examined on a single patient sample. Such panels have been identified for both breast and ovarian cancer11-13 and will soon be identified for other types of cancer as well. Two such tests are commercially available for the diagnosis of recurring breast cancer (Agendia’s MammaPrint, Genomic Health’s Oncotype DX Breast Cancer Assay),14 although they are highly complex and are performed in only a few clinical laboratories worldwide. Thus, there is an unmet need for multiplex (6) Philip, R.; Murthy, S.; Krakover, J.; Sinnathamby, G.; Zerfass, J.; Keller, L.; Philip, M. J. Proteome Res. 2007, 6, 2509–17. (7) Gnjatic, S.; Ritter, E.; Buchler, M. W.; Giese, N. A.; Brors, B.; Frei, C.; Murray, A.; Halama, N.; Zornig, I.; Chen, Y. T.; Andrews, C.; Ritter, G.; Old, L. J.; Odunsi, K.; Jager, D. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 5088–93. (8) Sasaroli, D.; Coukos, G.; Scholler, N. Biomark. Med. 2009, 3, 275–88. (9) Salama, O.; Herrmann, S.; Tziknovsky, A.; Piura, B.; Meirovich, M.; Trakht, I.; Reed, B.; Lobel, L. I.; Marks, R. S. Biosens. Bioelectron. 2007, 22, 1508– 16. (10) Bild, A. H.; Yao, G.; Chang, J. T.; Wang, Q.; Potti, A.; Chasse, D.; Joshi, M. B.; Harpole, D.; Lancaster, J. M.; Berchuck, A.; Olson, J. A., Jr.; Marks, J. R.; Dressman, H. K.; West, M.; Nevins, J. R. Nature 2006, 439, 353–7. (11) van ’t Veer, L. J.; Dai, H.; van de Vijver, M. J.; He, Y. D.; Hart, A. A.; Mao, M.; Peterse, H. L.; van der Kooy, K.; Marton, M. J.; Witteveen, A. T.; Schreiber, G. J.; Kerkhoven, R. M.; Roberts, C.; Linsley, P. S.; Bernards, R.; Friend, S. H. Nature 2002, 415, 530–6. (12) Dressman, H. K.; Berchuck, A.; Chan, G.; Zhai, J.; Bild, A.; Sayer, R.; Cragun, J.; Clarke, J.; Whitaker, R. S.; Li, L.; Gray, J.; Marks, J.; Ginsburg, G. S.; Potti, A.; West, M.; Nevins, J. R.; Lancaster, J. M. J. Clin. Oncol. 2007, 25, 517–25. (13) Bild, A. H.; Parker, J. S.; Gustafson, A. M.; Acharya, C. R.; Hoadley, K. A.; Anders, C.; Marcom, P. K.; Carey, L. A.; Potti, A.; Nevins, J. R.; Perou, C. M. Breast Cancer Res. 2009, 11, R55. (14) Dowsett, M.; Dunbier, A. K. Clin. Cancer Res. 2008, 14, 8019–26.
gene expression tests for cancer screening in POC settings.15 In addition, multiplex diagnostics assays have also been cleared by the FDA for the diagnosis of allergy, autoimmune disease, and cardiovascular disease,16 but again, these are only available in clinical laboratories and could profit from migration into a nearpatient setting. Microarray technology has clear advantages for multiplex assays and has been broadly embraced by the biomedical research community over the past 20 years for applications such as gene expression profiling and single nucleotide polymorphism (SNP) analysis. Such studies have significantly advanced our understanding of the genetic basis of disease.10,12,13,17 Several companies are developing multiplex molecular diagnostics assays based on conventional microarray technology, but only two (Roche’s AmpliChip CYP450 and Agendia’s MammaPrint) have been cleared by the FDA to date. The AmpliChip CYP450 test determines a patient’s genotype for different cytochrome P450 genes that impact drug metabolism,18 and the aforementioned MammaPrint test assesses the metastasis risk in breast cancer patients. These tests are presently available only in clinical laboratories because of their high complexity and dependence on expensive microarray laser scanners. Thus, there is a need for new microarray technology that is simple, inexpensive, and suitable for POC molecular diagnostics testing. Of note is a potentially “disruptive” microarray technology for detection of pathogen DNA without prior DNA amplification. This technology is based on DNA lithography in which DNA nanostructures are metalized to form nanowires.19 In particular, a target DNA duplex with sticky ends is captured by complementary oligonucleotide probes immobilized on two closely spaced electrodes. The captured target is then metalized to form a nanowire bridging the two electrodes, which completes the circuit between the two electrodes comparable to turning on a switch. Integrated Nano-Technologies is developing a platform technology (BioDetect) based on this technology for rapid detection of biodefense pathogens such as Bacillus anthracis.20 Their microarray sensors support both 14- and 16-assay formats, with plans for 250-assay chips. Microarray chips are housed within a disposable test card containing requisite microfluidics for sample preparation and metallization. Protein microarrays hold great promise as well, both in proteomics research and in multiplex diagnostics. In fact, Hartmann et al.16 recently made the point that protein microarrays are especially well suited for immunodiagnostics for several reasons, including their potential for highly multiplexed immunoassays, small sample size, minimal reagent requirements, and concentration sensitivity (signal proportional to concentration rather than mass). Several companies are developing multiplex immunodiagnostics assays based on microarray technology, but (15) Soper, S. A.; Brown, K.; Ellington, A.; Frazier, B.; Garcia-Manero, G.; Gau, V.; Gutman, S. I.; Hayes, D. F.; Korte, B.; Landers, J. L.; Larson, D.; Ligler, F.; Majumdar, A.; Mascini, M.; Nolte, D.; Rosenzweig, Z.; Wang, J.; Wilson, D. Biosens. Bioelectron. 2006, 21, 1932–42. (16) Hartmann, M.; Roeraade, J.; Stoll, D.; Templin, M. F.; Joos, T. O. Anal. Bioanal. Chem. 2009, 393, 1407–16. (17) Podder, M.; Ruan, J.; Tripp, B. W.; Chu, Z. E.; Tebbutt, S. J. BMC Med. Genomics 2008, 1, 5. (18) Jain, K. K. Mol Diagn 2005, 9, 119–27. (19) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998, 391, 775– 8. (20) Connolly, M. In Bionanotechnology: Global Prospects; Reisner, D. E., Ed.; CRC Press: Boca Raton, FL, 2009.
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only Randox’s Drugs of Abuse biochip has been cleared by the U.S. Food and Drug Administration (FDA) for their Evidence clinical laboratory analyzer. Thus, there is also a need for inexpensive, low-complexity proteomics array platform technologies suitable for near-patient testing. Several different multiplex assays based on optical biosensor technology have been described in the literature over the past decade that have the potential for addressing the aforementioned limitations in current POC tests and microarray technology, although few have yet reached the marketplace. These include label-free approaches such as reflectometric interference spectroscopy21 and surface plasmon resonance imaging,22-24 as well as label-based approaches such as chemiluminescence25-27 and fluorescence (including optical-fiber and planar waveguide biosensors).4,28-36 Although label-free approaches are less complex than label-based approaches, they are often less sensitive and thus less suitable for diagnostics applications requiring picomolar sensitivity or better. Fluorescence and chemiluminescence are both capable of subpicomolar detection, but each has its own strengths and weaknesses. Chemiluminescence is theoretically more sensitive because of its absence of background luminescence,37 but analytical sensitivity comparisons in which the two detection methods are compared side-by-side with the same analyte (or set of analytes) suggest that the two methods are roughly comparable (analytical sensitivity values within 10-fold of each other), with no clear preference for either.38-40 Thus, the choice between the two often depends on other factors such as the lower optical complexity of chemiluminescence (no light source or emission filters), the greater quantum yield of fluores(21) Proll, G.; Steinle, L.; Proll, F.; Kumpf, M.; Moehrle, B.; Mehlmann, M.; Gauglitz, G. J. Chromatogr. A 2007, 1161, 2–8. (22) Lausted, C.; Hu, Z.; Hood, L.; Campbell, C. T. Comb. Chem. High Throughput Screen. 2009, 12, 741–51. (23) Piliarik, M.; Vaisocherova, H.; Homola, J. Methods Mol. Biol. 2009, 503, 65–88. (24) Scarano, S.; Mascini, M.; Turner, A. P.; Minunni, M. Biosens. Bioelectron. 2010, 25, 957–66. (25) Heyries, K. A.; Loughran, M. G.; Hoffmann, D.; Homsy, A.; Blum, L. J.; Marquette, C. A. Biosens. Bioelectron. 2008, 23, 1812–8. (26) Deiss, F.; LaFratta, C. N.; Symer, M.; Blicharz, T. M.; Sojic, N.; Walt, D. R. J. Am. Chem. Soc. 2009, 131, 6088–9. (27) Karsunke, X. Y.; Niessner, R.; Seidel, M. Anal. Bioanal. Chem. 2009, 395, 1623–30. (28) Barzen, C.; Brecht, A.; Gauglitz, G. Biosens. Bioelectron. 2002, 17, 289– 95. (29) Pawlak, M.; Schick, E.; Bopp, M. A.; Schneider, M. J.; Oroszlan, P.; Ehrat, M. Proteomics 2002, 2, 383–93. (30) Tschmelak, J.; Proll, G.; Gauglitz, G. Biosens. Bioelectron. 2004, 20, 743– 52. (31) Taitt, C. R.; Anderson, G. P.; Ligler, F. S. Biosens. Bioelectron. 2005, 20, 2470–87. (32) Weissenstein, U.; Schneider, M. J.; Pawlak, M.; Cicenas, J.; EppenbergerCastori, S.; Oroszlan, P.; Ehret, S.; Geurts-Moespot, A.; Sweep, F. C.; Eppenberger, U. Proteomics 2006, 6, 1427–36. (33) Gorris, H. H.; Blicharz, T. M.; Walt, D. R. FEBS J. 2007, 274, 5462–70. (34) Ligler, F. S.; Sapsford, K. E.; Golden, J. P.; Shriver-Lake, L. C.; Taitt, C. R.; Dyer, M. A.; Barone, S.; Myatt, C. J. Anal. Sci. 2007, 23, 5–10. (35) Ligler, F. S. Anal. Chem. 2009, 81, 519–26. (36) Walt, D. R. Chem. Soc. Rev. 2010, 39, 38–50. (37) Roda, A.; Pasini, P.; Mirasoli, M.; Michelini, E.; Guardigli, M. Trends Biotechnol. 2004, 22, 295–303. (38) Sia, S. K.; Linder, V.; Parviz, B. A.; Siegel, A.; Whitesides, G. M. Angew. Chem., Int. Ed. 2004, 43, 498–502. (39) Rubina, A. Y.; Dyukova, V. I.; Dementieva, E. I.; Stomakhin, A. A.; Nesmeyanov, V. A.; Grishin, E. V.; Zasedatelev, A. S. Anal. Biochem. 2005, 340, 317–29. (40) Taieb, J.; Benattar, C.; Birr, A. S.; Lindenbaum, A. Clin. Chem. 2002, 48, 583–5.
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cent dyes (higher signal intensity), or the wider range of emission wavelengths available for fluorescent dyes (wavelength multiplexing).41 Also, most chemiluminescence assays are enzyme-linked, which results in higher assay complexity and slower reaction kinetics. Herein, we describe a new modality for performing rapid, inexpensive, multiplex diagnostics assays that is based on novel microarray chip technology that replaces expensive laser scanning with an intersecting grid of 100-µm-wide waveguides embedded in the chip’s substrate. Referred to as “in-plane parallel scanning” (IPPS), this technology is essentially a next-generation planar waveguide biosensor in which excitation and collection waveguides are microfabricated as a microarray on a silicon chip. It builds on previous planar waveguide biosensor approaches4,28-35 and recent advances in telecommunications to replace the glass substrate typically used in microarrays with a microfabricated one that can independently address tens to thousands of sensor elements on a single chip. In this article, we introduce the IPPS concept, describe its technological implementation, and report its proofof-principle validation in a low-density (two-analyte) multiplex immunoassay for human cytokine interleuken-1β (IL-1β) and Clostridrium difficile toxin A. The first is an inflammation and allergy biomarker, but is used clinically for diagnosing ventilatorassociated pneumonia, a nosocomial infection.42 The second is an enterotoxin implicated in antibiotic-associated nosocomial C. difficile infection.43 It is also used clinically for diagnosing such infections. These particular biomarkers were chosen because they are part of a wider panel of biomarkers that could be used in a POC microarray for diagnosing nosocomial infections in hospital settings. EXPERIMENTAL SECTION Reagents. Organic and Inorganic Chemicals. The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification: acetone (catalog no. 179124), dichlorodimethylsilane (catalog no. 440272), ethanol (absolute, catalog no. 459844), hydrogen peroxide (30%, catalog no. 216763), isopropyl alcohol (catalog no. 190764), phosphate buffered saline (PBS) (powder, pH 7.4, catalog no. P3813), sulfuric acid (catalog no. 320501), and toluene (anhydrous, catalog no. 244511). Immunochemicals. The following antibodies and antigens were ordered from BioLegend (San Diego, CA): Alexa Fluor 647 labeled antihuman IL-1β antibody (murine monoclonal; clone JK1B-1, affinity purified, catalog no. 508208), antihuman IL-1β antibody (murine, monoclonal; clone JK1B-2, affinity purified, catalog no. 508304), IL-1β (recombinant human, carrier-free, catalog no. 579402). The following antibodies and antigens were ordered from Meridian Life Science, Inc. (Saco, ME): anti-Clostridium difficile toxin A antibody (rabbit polyclonal, affinity purified, catalog no. B01245R) and anti-Clostridium difficile toxin A antibody (murine monoclonal; clone PCG4.1, Protein A purified, catalog no. C70517M). The latter was labeled with DyLight 649 using a (41) Chowdhury, M. H.; Aslan, K.; Malyn, S. N.; Lakowicz, J. R.; Geddes, C. D. J. Fluoresc. 2006, 16, 295–9. (42) Conway Morris, A.; Kefala, K.; Wilkinson, T. S.; Moncayo-Nieto, O. L.; Dhaliwal, K.; Farrell, L.; Walsh, T. S.; Mackenzie, S. J.; Swann, D. G.; Andrews, P. J.; Anderson, N.; Govan, J. R.; Laurenson, I. F.; Reid, H.; Davidson, D. J.; Haslett, C.; Sallenave, J. M.; Simpson, A. J. Thorax 2010, 65, 201–7. (43) Bartlett, J. G.; Gerding, D. N. Clin. Infect. Dis. 2008, 46 (Suppl 1), S12–8.
Figure 1. Schematic description of the IPPS-100 chip and the optical interrogation method. Light is coupled into the chip at the top left by a switching/scanning light source (1). The light propagates from left to right in a set of excitation waveguides (2), exciting fluorescence in the sensing wells (4) along a specific excitation waveguide. A major portion of the fluorescent light is coupled back into the chip and is guided by a set of collection waveguides (3) to the bottom edge of the chip, where it is detected by an external multielement detector (not shown).
Figure 2. Magnified photographs of the (a) IPPS-10 and (b) IPPS-100 chips. The IPPS-10 chip consists of a set of 10 parallel structures having a 500-µm pitch. Light couples to the S-shaped waveguide at the bottom of each pair and funnels into the longer waveguide that crosses the chip from left to right. The sensing wells are seen as brighter lines starting before the center of the chip. The different features of the IPPS-100 are described in Figure 1.
DyLight antibody labeling kit (Fisher Scientific, catalog no. PI53050) following the manufacturer’s instructions. Clostridium difficile toxin A toxoid was purchased from List Biological Laboratories, Inc. (Campbell, CA, catalog no. 153). Bovine serum albumin (1% w/v solution, catalog no. A3803) was purchased from Sigma-Aldrich. StabilCoat immunoassay stabilizer was purchased from SurModics (Eden Prairie, MN, product code SC01). IPPS Microarray Chips. The IPPS concept is illustrated in Figure 1. LioniX B.V. (Enschede, The Netherlands) fabricated the IPPS microarray chips using standard semiconductor chip manufacturing techniques such as photolithography, thin-layer deposition [low-pressure chemical vapor deposition (LPCVD) and plasma-enhanced chemical vapor deposition (PECVD)] and etching (reactive ion etching and wet etching). Silicon wafers (4-in.) were used as the initial substrate. A thin layer of Si3N4 was deposited on top of the silicon wafer, and the waveguides were defined using several photolithography and etching steps. A
top SiO2 layer was then deposited, generating the cladding layer for the waveguides. Finally, the sensing wells were defined and etched into the SiO2 layer. The 4-in. wafers were then diced into individual 10 mm × 10 mm chips. Two different chip designs were implemented. The IPPS-10 design contains a grid of 10 parallel 100-µm-wide waveguides used for both excitation and collection with a sensing well located on top of each (Figure 2a). The IPPS-100 design contains a 10 × 10 array of orthogonal excitation and collection waveguides with a sensing well located at each intersection point (Figure 2b). Both types of waveguides are 100 µm wide, thus creating 100 different 100-µm-square sensing wells. IPPS Chip Reader. Several generations and/or configurations of optical readers were built to interrogate and read the two different IPPS chip designs. All of these include a precise chip holder with tight tolerances for aligning the chip to both light source and photodetector. In early versions, a 642-nm laser diode Analytical Chemistry, Vol. 82, No. 21, November 1, 2010
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(StockerYale, Inc., Lasiris PureBeam) generated 100-µs pulses of light that were coupled to the chip through a fast optical switch (LIGHTech Fiberoptics, Inc., LT-900). Collected light was coupled out of the chip to a second optical switch and then directed to a photomultiplier tube (PMT) module (Hamamatsu, H5784-20) through a band-pass filter (Omega Optical, 670DF0) that rejected exciting light. In our second-generation reader, we replaced the laser source and first optical switch with a low-cost, miniature 10laser-diode chip. The second optical switch and PMT module were replaced with a single linear charge-coupled device (CCD) array (Hamamatsu, S10420-1006) with integrated miniature collimating optics and band-pass filters. Both modules were developed and customized for us by AVO Photonics (Horsham, PA). All systems include temperature control of the chip using a thermo-electric cooler (TEC, Melcor, SH1.0-23-06 L) unit mounted right underneath the chip with closed-loop control. The entire system control, data collection, and storage was done automatically using a Data Acquisition (DAQ) unit (National Instruments, NI USB 6251) and proprietary LabView (National Instruments) based software and user interface. Cleaning and Functionalization of IPPS Chips. IPPS-10 chips were initially cleaned in organic solvents and piranha solution and then hyrdophobized with dichlorodimethylsilane (DDS). All cleaning steps were carried out in a fume hood or vacuum oven. Piranha solution was prepared by adding 35 mL of 95-98% sulfuric acid to a beaker placed in an ice bath and then slowly adding 30 mL of 30% hydrogen peroxide dropwise so that the solution did not boil. DDS solution (ca. 10% v/v) was prepared by adding 6 mL of 99.5% DDS to 54 mL of anhydrous toluene. The organic cleaning steps involved initially rinsing new chips with acetone from a squeeze bottle to remove visible debris and then immersing them in an acetone bath placed in an ultrasonic cleaner for 3 min at room temperature with a 50% ultrasound duty cycle (30 s of ultrasound for each minute of immersion). The ultrasonic cleaning step was repeated four times, twice with fresh acetone and twice more with fresh isopropanol. Chips were then rinsed five times with ultrapure water (18 MΩcm) and dried with dry N2 gas. Piranha cleaning involved immersion in a freshly prepared piranha solution placed in an ultrasonic cleaner for 20 min at room temperature with 20% ultrasound duty cycle during the last 10 min. Chips were again rinsed five times with ultrapure water (18 MΩ cm) and initially dried with dry N2 gas, followed by at least 30 min of additional drying in a vacuum desiccator. DDS silanization was based on a procedure described previously.44 In brief, chips were first immersed in DDS solution for 30 min at room temperature with gentle agitation, after which they were dip rinsed three times in absolute ethanol, quickly dried with dry N2 gas, and cured at 120 °C for 60 min in a vacuum oven flushed with three exchanges of dry N2 gas. DDS-coated chips were stored in a vacuum desiccator until use. Antibody Immobilization onto IPPS Chips. Two of us (J.N.H. and D.A.C.) previously investigated several different methods for immobilizing antibodies onto silica chips, including physical adsorption, coupling via a biotin-avidin bridge, and
covalent coupling either directly to silica surfaces or through a polymeric spacer layer.44-46 Physical adsorption and biotin-avidin bridging were clearly superior to the other methods, and biotin-avidin bridging exhibited slightly higher antigen-binding capacities and somewhat lower nonspecific binding (NSB) than physical adsorption. However, the methodology for biotin-avidin bridging is also more complex than physical adsorption, with more possible points of failure. For this reason, we decided that physical adsorption to DDS-silanized IPPS-10 chips would be satisfactory for the proof-of-principle studies reported in this article and would also be easier to debug in case immobilization problems were encountered. Nevertheless, we plan to reexamine biotin-avidin bridging in future studies. Antibodies were patterned on DDS-coated IPPS-10 chips using a coating gasket affixed to the sensing side of the chip. J.N.H. and D.A.C. previously used this method to pattern up to three different antibodies onto a single planar waveguide sensor, so we thought it suitable for the duplex immunoassay reported herein. Two different types of gaskets were custom fabricated from silicone rubber by ALine, Inc. (Rancho Dominguez, CA). One contained a single interior compartment (5.5 mm × 5.5 mm × 2 mm, 61-µL volume) allowing immobilization of the same capture antibody to all 10 sensing wells, whereas the other was subdivided into two interior compartments (each 5.5 mm × 2.25 mm × 2 mm, 25-µL volume) allowing two different antibodies to be patterned in adjacent areas of the same chip. Each area contained four sensing wells (the interior rib of the coating gasket covered the other two). Both types of gaskets had exterior dimensions of 7.5 mm × 7.5 mm × 2 mm and were coated on one side with a thin layer of contact adhesive. We used a murine monoclonal antibody (Biolegend no. 508304) as the capture antibody for the IL-1β assay and a rabbit polyclonal antibody (Meridian no. B01245R) as the capture antibody in the C. difficile toxin A assay. Based on previous experience,44 we initially added 125 µg/mL (833 nM) capture antibody in PBS to the coating gasket and incubated for 3 h at room temperature. Chips used in the IL-1β analytical sensitivity determination were coated under these conditions. Later, we determined that concentrations down to 12.5 µg/mL (83 nM) and incubation times as short as 15 min at room temperature gave results comparable to those obtained under our initial immobilization conditions. Based on these results, all subsequent immobilizations used the following conditions: 12.5 µg/mL capture antibody and 30-min incubation time at room temperature. After immobilization, antibody solution was removed from the gasket, and the chip was rinsed five times in PBS. Different blocking protocols were used depending on the storage conditions. Chips for immediate use were blocked using StabilCoat immunoassay stabilizer for 30 min at room temperature. Those for long-term storage were blocked with StabilCoat for 4 h at room temperature and then stored at 4 °C until use. Immunoassays. A one-compartment gasket was affixed to the sensing surface of an IPPS-10 chip for placing the sample solution to be analyzed. The chip was then inserted into the reader, and
(44) Herron, J. N.; Wang, H.-K.; Janatova´, V.; Durtschi, J. D.; Christensen, D. A.; Caldwell, K. D.; Chang, I.-N.; Huang, S.-C. In Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; Vol. 110, pp 115163.
(45) Huang, S.-C.; Caldwell, K. D.; Lin, J.-N.; Wang, H.-K.; Herron, J. N. Langmuir 1996, 12, 4292–4298. (46) Lin, J.-N.; Chang, I.-N.; Andrade, J. D.; Herron, J. N.; Christensen, D. A. J. Chromatogr. A 1991, 542, 41–54.
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the immunoassay was performed. All assays had at least the following three mandatory steps: Step 1 was the negative control, in which a 40-µL volume of tracer solution consisting of the specific labeled tracer antibody (250 ng/mL) and at least one nonspecific labeled antibody (same concentration) in the assay matrix (40 mg/mL BSA mixed 50%/ 50% with PBS) was placed in the gasket. Background fluorescence data were collected every 10 s for 2-5 min, after which the tracer solution was removed. Step 2 was the target assay, in which a 40-µL volume of the target analyte in the above tracer solution was added to the gasket. The binding of the target and complex formation on the chip surface were monitored through the increase in the fluorescence signal measured every 10 s for 5 min, after which the tracer solution was removed. Step 3 was the positive control, in which a 40-µL volume of high-concentration (usually 100 pM) target analyte in tracer solution was added to the gasket. The binding of the target and complex formation on the chip surface were monitored through the increase in the fluorescence signal collected every 10 s for 2-5 min. Data Analysis. To fully benefit from the IPPS platform’s ability to monitor molecular binding and complex formation in real time, we analyzed binding rate data rather than mid- or end-point fluorescence intensity measurements. This analytical approach has been described previously for capillary and planar waveguide fluorescence biosensors.4,47-52 Kinetic assays offer a clear advantage over end-point assays in terms of assay time because solidphase antigen-antibody binding reactions can take an hour or more to reach equilibrium. In addition, kinetic assays often have higher sampling rates than end-point assays, offering some precision advantages as well. Data analysis proceeded in three steps: (1) background correction of fluorescence intensity data, (2) channel correction of fluorescence intensity data, and (3) rate determination. (1) Background correction redefines the average of the negative control data (normally flat) for a given channel as the zero point for that data set. This is accomplished by subtracting the average of the negative control data for a given channel from each and every data point in the same channel. (2) Channel correction uses the average positive control signal across all channels as a fiduciary to correct for small channel-tochannel intensity variations that might arise from optical coupling variations or from nonuniformities in the capture antibody layer. In particular, positive control data collected in each channel for a given time point were regressed against the all-channel average for that same time point. Then, all of the data collected from a given channel were divided by regression coefficient for that channel. (47) Badley, R. A.; Drake, R. A. L.; Shanks, I. A.; Smith, A. M.; Stephenson, P. R.; Thomas, J. D. R. Philos. Trans. R. Soc. London B 1987, 316, 143–60. (48) Daniels, P. B.; Fletcher, J. E.; O’Neill, P. M.; Stafford, C. G.; BacareseHamilton, T.; Robinson, G. A. Sens. Actuators B 1995, 27, 447–51. (49) Duveneck, G. L.; Pawlak, M.; Neuscha¨fer, D.; Ba¨r, E.; Budach, W.; Pieles, U.; Ehrat, M. Sens. Actuators B 1997, 38, 88–95. (50) Misiakos, K.; Kakabakos, S. E. Biosens. Bioelectron. 1998, 13, 825–30. (51) Hofmann, O.; Voirin, G.; Niedermann, P.; Manz, A. Anal. Chem. 2002, 74, 5243–50. (52) Gauglitz, G. Anal. Bioanal. Chem. 2005, 381, 141–55.
(3) Analyte binding rates were determined for each channel using corrected target assay data. In most cases, fluorescence versus time data were linear over the 5-min target assay period, and rates were determined by linear regression using Excel’s Slope function. For concave-downward curves, the initial rate was determined either from a truncated data set or by nonlinear leastsquares using a reactant depletion kinetic model.4 It should be mentioned that the positive control data can also be used as a fiduciary to calibrate IPPS chips used in POC settings to the same assay performed in the reference laboratory. Although this possibility is not explored herein, we plan to examine it in future studies. SAFETY CONSIDERATIONS The piranha solution used for cleaning IPPS chips is both a strong acid and a potent oxidizing agent. Furthermore, its preparation involves addition of 30% hydrogen peroxide to nearly pure sulfuric acidsa strongly exothermic reaction. For these reasons, all operations involving piranha preparation or use were carried out in a fume hood by trained personnel wearing appropriate laboratory protective equipment. Sulfuric acid was first added to a beaker and chilled to 4 °C in an ice bath. The hydrogen peroxide was slowly added dropwise with gentle stirring to prevent boiling. Each batch of solution was used only once for cleaning IPPS chips (also in the fume hood) and then properly disposed by institutional environmental health and safety personnel. RESULTS AND DISCUSSION IPPS Chips: Concept, Design, Optical Simulations, and Attributes. Two different optical schemes are typically used for reading conventional microarrays: laser scanning and optical imaging. The former scans the microarray in increments of a few micrometers using confocal optics with a laser source and photomultiplier tube detector, whereas the latter images the entire microarray at one time using a broad-spectrum light source and charge-coupled device (CCD) camera.53 In contrast, IPPS microarrays are scanned by manipulating light within the chip itself, guiding it to each and every sensing zone. Different microarray rows are selected through a fast switching/scanning light source that butt-couples to the chip and sequentially directs the light to every row of the array (Figure 1, feature 1). Light is collected at the other end of the chip using a detector array measuring all columns in parallel. The 10-channel IPPS-10 chip contains 10 100 µm × 10000 µm primary waveguides spaced on 500-µm centers across the chip (Figure 2a). An 80 µm × 4500 µm sensing well is microfabricated on top of each. In addition, each primary waveguide has an associated short “S-shaped” secondary waveguide located just below its left edge (see Figure 2a) that is used for light coupling. In particular, exciting light is coupled into the secondary waveguide and funneled into the primary, where it propagates by total internal reflection from left to right, setting up an evanescent field along the entire length of the sensing well. The field penetrates about 70 nm into the sensing well (Figure 3b), where it excites fluorescently tagged analyte molecules captured by recognition molecules immobilized in the sensing well. A portion of the anisotropic fluorescence emission then favorably back-couples into (53) Scha¨ferling, M.; Nagl, S. Anal. Bioanal. Chem. 2006, 385, 500–17.
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Figure 3. Optical simulations of electromagnetic wave amplitude within a slab waveguide and light scattering in a water-filled sensing well. (a) Collection efficiency in the TE and TM modes of a slab waveguide for randomly oriented dipoles placed 20 nm from the waveguide surface versus thickness of the silicon nitride waveguide layer. (b) Theoretical penetration depth (i.e., distance at which the electromagnetic wave amplitude decays to 1/e of the maximum value) into a well filled with water (n ) 1.33) as a function of the nitride waveguide thickness. (c) Two-dimensional representation of the electromagnetic field (absolute value) where the evanescent field of the light propagating in an excitation waveguide from left to right “hits” the refractive-index discontinuity associated with the front end (x ) 0, z ) 1 µm) of the sensing well filled with water (n ) 1.33). This discontinuity induces some light scattering mainly upward and downward. The amount of scattering depends on the well profile created in the etching step. The steeper the walls, the larger the scattering.
the primary waveguide,54-56 which channels it to a photodetector located at the left side of the chip. The more complex 100-channel IPPS-100 chip requires a different design in which two intersecting sets of waveguides are embedded in the chip’s substrate, creating (54) Polerecky, L. u.; Hamrle, J.; MacCraith, B. D. Appl. Opt. 2000, 39, 3968– 77. (55) Baldini, F.; Carloni, A.; Giannetti, A.; Porro, G.; Trono, C. Anal. Bioanal. Chem. 2008, 391, 1837–44. (56) Baldini, F.; Bolzoni, L.; Giannetti, A.; Kess, M.; Kramer, P. M.; Kremmer, E.; Porro, G.; Senesi, F.; Trono, C. Anal. Bioanal. Chem. 2009, 393, 1183– 90.
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a mesh-like structure (Figures 1 and 2b). One set is used to launch the excitation light (Figure 1, feature 2), and the other is used to collect fluorescent emission (Figure 1, feature 3). An etched well at each intersection point defines the sensing zone where specific recognition molecules are immobilized (Figure 1, feature 4). The IPPS-10 chip is well suited for low-degree multiplexing where 10 or fewer tests are run simultaneously on the same sample. For this reason, it was used for the monoplex and duplex immunoassays reported herein. The IPPS-100 chip is better suited for medium-degree multiplexing such as the gene expression applications mentioned in the introduction. Such applications are currently under investigation and will be reported separately. Extensive optical simulations were performed in three iterations during IPPS chip design and optimization. Simulations were based on the work of Hoekstra and Elrofai57 and beam propagation methods.58 The first step was to predict the optical efficiency of IPPS-10 and IPPS-100 chips by simulating the effects of both waveguide thickness and the spacing of randomly oriented emitting dipoles off the waveguide on excitation and collection efficiency. Results of the latter simulation (see Figure 3a) suggested that light radiation from the emitting dipoles is anisotropic in nature, with 50% or more coupling back into waveguides with thicknesses in the range of 100-150 nm. This result is consistent with an optical theory developed by Polerecky et al.54 describing radiation emitted by dipoles located on top of a waveguide layer. Next, we focused on optimization of different design parameters such as waveguide material and thickness to provide the necessary penetration depth for the evanescent wave (Figure 3b) while minimizing the light scattering that adds to the noise and to the channel-to-channel variations. In addition, sensing well structure and etching process were analyzed and modified to reduce light scattering (Figure 3c) and provide better surface properties for attaching the capture molecules. Also, waveguide structure at the chip edges was optimized using lateral and vertical tapering technologies to increase the in and out light coupling efficiencies and robustness (the IPPS-10 chips employ both of these tapering technologies as shown in Figure 2a). IPPS has the following advantages over existing microarray systems: Multiplexing. By immobilizing different probes to different wells, we can multiplex a large number of tests on a single chip (Figure 2). This enables us to run either multiplex tests on a single sample or, when combined with sample delivery microfluidics, multiplex tests on multiple samples. Small Scanner Footprint. Because scanning takes place within the chip, the reader can be compact because of the simplicity of its optics (compared to a laser scanner) and the small number of required components. This lends itself to applications where portable desktop or handheld devices are required. Reaction Kinetics Monitoring. Fast optical switching during excitation and parallel collection of fluorescence allows scanning of the entire chip in seconds, allowing real-time monitoring of reaction kinetics of biochemical processes taking place within the wells (see Figure 4), as opposed to measuring the reaction end point as done with laser scanning. This enables more data points (57) Hoekstra, H. J. W. M.; Elrofai, H. B. H. Phys. Rev. E 2005, 71, 046609. (58) Hoekstra, H. Opt. Quantum Electron. 1997, 29, 157–71.
Figure 4. IPPS-10 chip fluorescence intensity response for a twoanalyte multiplex immunoassay. Capture antibodies for C. difficile toxin A and human IL-1β were immobilized in channels 1-4 and 7-10, respectively. After being coated, the chip was fitted with a onecompartment assay gasket that exposed all 10 channels to the same sample. The following series of controls and analytes were sequentially added and removed from the gasket: Sample 1, tracer antibodies and BSA matrix; sample 2, 10 pM toxin A toxoid, tracer antibodies, and BSA matrix; sample 3, BSA matrix without tracer antibodies; sample 4, 10 pM IL-1β, tracer antibodies, and BSA matrix; sample 5, BSA matrix without tracer antibodies; sample 6, 100 pM each toxin A toxoid and IL-1β, tracer antibodies, and BSA matrix. Sample buffer and tracer antibodies are described in the footnotes to Table 1. The fluorescence intensity for each channel was corrected for both background and channel before plotting. The signals for the four toxin A channels were offset by 20 mV so that the baseline responses of both assays could be clearly seen.
to be collected in a much shorter time (
