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An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Cryptosporidium parvum in water has been developed. The targ...
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Anal. Chem. 2003, 75, 3890-3897

Immobilized Enzyme-Linked DNA-Hybridization Assay with Electrochemical Detection for Cryptosporidium parvum hsp70 mRNA Zoraida P. Aguilar† and Ingrid Fritsch*

Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701

An electrochemical enzyme-linked immobilized DNAhybridization assay for the detection of Cryptosporidium parvum in water has been developed. The target molecule was a 121-nucleotide sequence from the C. parvum heat shock protein 70 (hsp70 mRNA from U71181 gene). This analyte offers the possibility of distinguishing dead from live oocysts. The assay involves covalent attachment of a primary DNA probe via its 5′-amine-terminus to selfassembled monolayers of mercaptoundecanoic acid to a gold surface. The primary DNA probe was used to capture the target (sequence 1039-1082 of U71181gene for the mRNA), by hybridization to a 20-base complementary sequence on the target (at sequence 1063-1082). A secondary DNA probe labeled with alkaline phosphatase (AP) was then hybridized to base sequence 1039-1062 on the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution, in which gold-coated silicon wafer modified with the complete assembly of the assay components was incubated, is linear with concentration of the target (550 µg/mL, where P1 and P2-AP concentrations are 50 µg/mL). A detection limit of 2 µg/mL (or 146 nM) of the DNA target was obtained. Cross-reactivity tests showed high selectivity for heat-shocked C. parvum. No signal was obtained for either the synthetic DNA for hsp70 of Campylobacter lari, Escherichia coli, Giardia lamblia, Salmonella typhimurium, and Listeria monocytogenes or for the products of heat-shocked whole organisms of E. coli, G. lamblia, Staphylococcus aureus, and Cryptosporidium muris. We report a new assay for the detection of Cryptosporidium parvum that is selective, capable of distinguishing dead from live oocysts, and suitable for miniaturization and array formats. The approach is based on an electrochemical detection scheme for mRNA for heat shock protein 70 (hsp70). The work presented here demonstrates the concept on a macroscale and makes * To whom correspondence should be addressed. Tel.: (479) 575-6499. Fax: (479) 575-4049. E-mail: [email protected]. † Current address: Department of Chemistry, Rm. 105 Crosley Tower, University of Cincinnati, Cincinnati, OH 45221-0172. Telephone: (513) 556-9203E-mail: [email protected].

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predictions for the small scale based on our previous work,2 which indicates that high speed and sensitivity are also achievable for such assays when downsized. The detection of C. parvum, the 4-5-µm oocyst3 excreted through the feces of the infected host that is resistant to ordinary water treatment processes (i.e., chlorination, ozonation, and ordinary filtration processes),4 is important because of its widespread impact on human health. The oocyst production by infected hosts that reaches 109-1010 in 24 h, ensures a high level of contamination in the environment that favors waterborne transmission.4 Of the known species of the genus C. parvum is claimed as the one responsible for diseases in humans and domestic animals.3 Between 250 and 500 million infections of C. parvum occur annually in Asia, Africa, and Latin America,4 and no specific drug treatment exists for cryptosporidiosis.4 C. parvum is claimed as a major pathogen contributing to the deaths of 11 000 children each day and the deaths of 5 million each year worldwide by causing diarrheal illness.5 The massive outbreak experienced in Milwaukee, WI, in 1993 that affected 403 000 individuals was judged to cost the community some $53 million in lost wages, lost productivity, medical bills, and emergency room visits and $100 million in claims for loss of life.4 The number of oocysts generally found in environmental water is low, but an infectious dose is suggested to be as low as 30 oocysts,6 and a 0.5% distinct probability of infection has been calculated for ingestion of a single oocyst.7 Unfortunately, the current EPA approved methods of detection (methods 1622 and 1623) can only detect >100 oocysts/L with a high number of false positives and false negatives. In addition, those methods are slow (2-3 weeks), expensive ($250-500),8 require experienced staff, and are unable to distinguish between viable and nonviable oocysts.5,8-13 The major steps involve filtration, (1) Mead, J., R.; Bonafonte, M. T.; Arrowood, M. J. On the National Center for Biotechnology Information, 1996; http://www.ncbi.nlm.nih.gov/, accessed March 28, 2002. (2) Aguilar, Z. P.; Vandaveer, W. R.; Fritsch, I. Anal. Chem. 2002, 74, 33213329. (3) Current, W. L. ASM News 1988, 54, 605-611. (4) Smith, H. V. R., J. B. Parasitol. Today 1998, 14, 14-22. (5) Newman, A. Anal. Chem. 1995, 67, 1A-734A. (6) Dupont, H. L.; Chappell, C. L.; Sterling, C. R.; Rose, J. B.; Jakubowski, W. N. Engl. J. Med. 1995, 332, 855-859. (7) "Protozoan parasites (Cryptosporidium, Giardia, Cyclospora). In Guidelines for Drinking Water Quality; Addendum: Microbiological agents in drinking water; World Health Organization, 2002 (http://www.who.int/water_sanitation_health/GDWQ/Microbiology/ Microbioladd/microadd5.htm). (8) Allen, M. J.; Clancy, J. L.; Rice, E. W. J. AWWA 2000, 92, 64-76. (9) Atherholt, T. B.; Korn, L. R. J. AWWA 1999, 91, 95-102. (10) Erickson, B. Anal. Chem. 1998, 70, 49A-52A. 10.1021/ac026211z CCC: $25.00

© 2003 American Chemical Society Published on Web 06/21/2003

immunomagnetic recovery of the oocysts from the filters, immunofluorescence assay for quantifying the oocysts, and dye staining with differential interference microscopy detection for confirmation of the assay.5,10-13 A number of molecular techniques (a technique that detects molecules rather than the entire organism, an organelle, or a fragment from the organism), for detection of C. parvum have been published recently.14,15 Fluorescence in situ hybridization method targeting an rRNA in C. parvum has also been reported.16 However, the presence of rRNA is not an indicator of oocyst viability.10 In vitro cell culture is another test for viability, but this method cannot be performed in a water treatment facility routinely or easily.17 Messenger RNA (mRNA) is generated only in living organisms, but the lifetime of mRNA is often only a few seconds to a few minutes.18 This makes mRNA an optimal target molecule to distinguish viable from nonviable organisms.13,19,20 The hsp70 mRNA, in particular, is produced only by viable organisms upon applying heat shock and can therefore be used for molecularlevel amplification in the detection of a single organism. Hence, we have chosen a hsp70 mRNA sequence specific to C. parvum oocysts as the target sequence in this study.13 Recently, Durst and co-workers developed electrochemiluminescence (ECL) and fluorescence methods to detect mRNA for hsp 70, extracted from C. parvum in water that had been concentrated via filtration, separated via immunomagnetic beads, and amplified with nucleic acid sequence-based amplification.13,19,20 However, in those methods, sample preparation and amplification are time-consuming. Detection of photons is hindered by turbid solutions that scatter the light and any absorbing or reflecting materials that stand between photon generation and detection sites (e.g., such as container materials), so that the design of those systems must take these issues into consideration. In addition, because ECL involves photon detection, it requires more instrumental components than electrochemistry alone. To our knowledge, only Wang and co-workers21,22 have reported the use of electrochemical detection with DNA hybridization for detection of Cryptosporidium. That approach21,22 involves adsorptive immobilization of the probe DNA on the carbon paste transducer for the capture and subsequent detection of an rRNA oligonucleotide that is specific to C. parvum. The reproducibility of the response is limited by the efficiency of the hand-mixing process (11) Newman, R. D.; Jaeger, K. L. W. T.; Lima, A. A.; Guerrant, R. L.; Sears, C. L. J. Clin. Microbiol. 1993, 31, 2080-2084. (12) Clancy, J. L.; Bukhari, Z.; McCuin, R. M.; Matheson, Z.; Fricker, C. R. J. AWWA 1999, 91, 60-68. (13) Bauemner, A. J.; Humiston, M. C.; Montagna, R. A.; Durst, R. A. Anal. Chem. 2001, 73, 1176-1180. (14) Xiao, L.; Alderisio, K.; Limor, J.; Royer, M.; Lal, A. A. Appl. Environ. Microbiol. 2000, 66, 5492-5498. (15) Xiao, L.; Singh, A.; Limor, J.; Graczyk, T. K.; Gradus, S.; Lal, A. Appl. Environ. Microbiol. 2001, 67, 1097-1101. (16) Vesey, G.; Ashbolt, N.; Fricker, E. J.; Deere, D.; Williams, K. L.; Veal, D. A.; Dorsch, M. J. Appl. Microbiol. 1998, 85, 429-440. (17) Current, W. L.; Garcia, L. S. Clin. Microbiol. Rev. 1991, 4, 325-358. (18) Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman and Co.: New York, 1988. (19) Esch, M. B.; Locascio, L. E.; Tarlov, M. J.; Durst, R. A. Anal. Chem. 2001, 73, 2952-2958. (20) Esch, M. B.; Baeumner, A. J.; Durst, R. A. Anal. Chem. 2001, 73, 31623167. (21) Wang, J.; Rivas, G.; Parrado, C.; Cai, X.; Flair, M. N. Talanta 1997, 44, 2003-2010. (22) Wang, J.; Fernandes, J. R.; Kubota, L. T. Anal. Chem. 1998, 70, 36993702.

Figure 1. Cross section schematic of an immobilized, enzymelinked DNA-hybridization assay for C. parvum. The modified gold macrochip is separate from the bare gold detecting electrode. See text for details.

of preparing the carbon paste electrode, nonspecific adsorption that is related to probe adsorption time, the use of blocking agents to minimize nonspecific adsorption, the pretreatment process of the carbon paste electrode that gives a well-defined peak in the absence of the probe, and the displacement of the adsorbed probe after hybridization longer than 7 min. The approach described here is also a DNA-hybridization assay with electrochemical detection (Figure 1) that introduces several new features and may potentially solve many of the problems that were previously reported. First, the assay detects hsp70 mRNA of C. parvum (and the DNA gene for mRNA), providing a way to detect viable organisms. The hsp70 mRNA can be amplified by subjecting the oocyst to heat shock and may allow detection of low concentrations of the oocyst. Second, immobilization involves not only covalent attachment (through amide formation with selfassembled monolayers (SAMs) of 11-mercapto-1-undecanol (MUA) on gold) but also a location that is separate from the detecting electrode (no electron-transfer events through the modifying layer), improving stability of the modified surface. Third, the detecting electrode is bare (because it is separate from the modified surface), allowing a larger signal to be observed than if it were modified with the assay components itself. Fourth, the assay is based on hybridization of complementary sequences of a primary probe (P1), the target (T), and secondary probe (P2) previously described,23 but uses alkaline phosphatase (AP) conjugated to P2 to produce an electrochemically active species (paminophenol, PAPR, from p-aminophenyl phosphate, PAPP) for detection at the electrode that amplifies with time, as opposed to (23) DeRiemer, L. H.; Meares, C. F. Biochemistry 1981, 20, 1606-1612.

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intercalation of electroactive molecules or direct detection of guanine residues. Finally, we have designed the assay chemistry so that it may be easily incorporated with our miniaturized device in the future,24-26 with the intention to drastically improve assay time and detection limits, as we have previously demonstrated for electrochemical detection of IgG using microcavity devices.2 EXPERIMENTAL SECTION Chemicals and Materials. All chemicals were reagent grade and used as received, unless otherwise indicated. Tris(hydroxymethyl)aminomethane (Tris), Tris-HCl (1 M Tris, pH 8.0, DNase and RNase free), sodium citrate, ethylenediaminetetraacetic acid (EDTA), 2-chloroacetamide, bovine serum albumin fraction V powder (BSA), alkaline phosphatase, 1-ethyl-3-(3-dimethylamino)propyl carbodiimide hydrochloride (EDC), lithium chloride anhydrous, sodium azide, and p-aminophenol hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO). Sodium dodecyl sulfate (SDS) was obtained from J. T. Baker (Philipsburg, NJ). Potassium chloride was obtained from Aldrich (Milwaukee, WI). Absolute ethanol (200 proof) was obtained from AAPER (Shelbyville, Kentucky). PAPP was synthesized, purified, and characterized as previously described by Aguilar et al.2 Aqueous solutions were prepared using high-purity deionized (DI) water from a Millipore Milli-Q filtration system (model RG, Bedford, MA). All oligonucleotides used in this study were purchased from Genemed Synthesis, Inc. (San Francisco, CA) and Invitrogen Life Technologies (Grand Island, NY). The primary probe, P1, is a 33base oligonucleotide (5′-TAT AGG GAG AAG GTA GAA CCA CCA ACC AAT ACA-3′, 10 921 g/mol) containing a 20-base sequence that is complementary to the target DNA (T) and a C6 amino linker at the 5′-end that serves as a spacer from the macrochip. The secondary probe, P2-AP, consists of a 42-base oligonucleotide labeled with alkaline phosphatase at the 5′-end (5′-ACA TCA TGT ACA GAT CTC TTG TCC CGC AAC TAC GAA GGT CTG-3′, 12 834 g/mol). It has 24 bases complementary to the 3′-end of the target DNA. The target DNA (5′-3′ sequence 1039-1082 of U71181: 5′-GGA CAA GAG ATC TGT ACA TGA TGT TGT ATT GGT TGG TGG TTC TA-3′, 13 704 g/mol)1 is a 44-base oligomer that is specific for C. parvum hsp70 mRNA based on a previously published study.13 The target sequence 1063-1082 is complementary to P1 while 1039-1062 is complementary to P2-AP. Oligonucleotides for hsp70 of Escherichia coli and hsp60 of Campylobacter lari and heat shock proteins of Giardia lamblia, Listeria monocytogenes, and Salmonella typhi were tested for crossreactivity. The oligonucleotide sequences were chosen with the maximum region of homology to the hsp70 mRNA gene of C. parvum based on the published sequences.27 The C. parvum oocysts at a concentration of (0.26 ( 0.05) × 106 oocysts/mL (heat shocked for 10 min at 43 °C by the supplier and heat shocked for additional 10 min at 43-45 °C in our laboratory), Cryptosporidium muris oocysts at a concentration of (0.26 ( 0.05) × 106/mL (heat shocked for 10 min at 43 °C by the supplier and heat shocked for additional 10 min at 43-45 °C in our laboratory) and G. lamblia (24) Henry, C.; Fritsch, I. Anal. Chem. 1999, 71, 550-556. (25) Henry, C.; Fritsch, I. J. Electrochem. Soc. 1999, 3367-3373. (26) Vandaveer, I., W. R. Ph.D. Dissertation, University of Arkansas, Fayetteville, 2002. (27) NCBI.; NLM.; NIH National Center for Biotechnology Information Sequence Viewer, http://www.ncbi.nih.gov, NCBI, September 24, 2001.

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cysts at a concentration of (0.26 ( 0.05) × 106/mL (heat shocked for 10 min at 43 °C by the supplier and heat shocked for additional 10 min at 43-45 °C in our laboratory) were obtained from Waterborne, Inc. (New Orleans, LA). The Escherichia coli and the Staphylococcus aureus were courtesy of the Department of Microbiology. The mRNA from heat shocked E. coli was isolated and detected using a kit (Tri Reagent and FORMazol, Molecular Research Center, Inc., Cincinnati, OH). A gold coin (Canadian Maple Leaf, 99.99%) and a chromiumplated tungsten rod (R. D. Mathis, Long Beach, CA) served as sources for metal deposition. Silicon wafers (125 mm diameter and 365-406 µm thick) with crystal orientation of (100) were obtained from Wacker Siltronic Corp. (Portland, OR). Polyimide (PI-2721, HD MicroSystems, Du Pont) was used according to DuPont specifications. Dialysis bags and Spectra/Gel absorbent were obtained from Spectrum Laboratories, Inc (Compton, CA). Ar gas and liquid N2 (nitrogen refrigerated liquid UN 1977) were obtained from PG Walker (Springdale, AR). Glovebags were obtained from Instruments for Research and Industry (Cheltenham, PA). All other chemicals and materials were from VWR. Buffer Solutions. The buffer solutions used in this study are as follows: (a) PBS, 0.1 M KH2PO4 and 6.2 mM Na2HPO4 in 14.3 mM NaCl at pH 6.0; (b) 20× SSC/BSA, 1 mg/mL BSA, 0.5 mg/ mL 2-chloroacetamide, 173.5 g/L NaCl, 88.2 g/L sodium citrate, 200 mg/L SDS, and 200 mg/L sodium azide, pH 7.6 (adjusted with 6 M HCl or 6 M NaOH); (c) 10 mM TE buffer, 10 mM TrisHCl, 1 mM EDTA, pH 8.0; (d) 0.1 M Tris, 0.10 M Tris, 1 mM magnesium chloride, and 0.02% (w/v) sodium azide, pH 9.0 (adjusted with 6 M HCl or 6 M NaOH). Caution: Wear gloves when using NaN3 because it is a carcinogenic agent! Fabrication of Macrochips. Au macrochips (∼1.4 cm × ∼1.2 cm for electrodes, where the electroactive area is about 0.6-1 cm2, and 1.2 cm × 1.2 cm for surface modification) were made from a 125-mm-diameter silicon wafer substrate that had 1.4-1.8 µm of SiO2 deposited on both sides at 250 °C by plasma-enhanced chemical vapor deposition (Plasma Therm System VII). Deposition of a 15-Å adhesion layer of Cr and 1000 Å of Au was carried out using an Edwards Auto 306 Turbo thermal evaporator (Edwards High Vacuum Instrument International, West Sussex, U.K.) to fabricate Au macrochips. Polyimide macrochips (1.2 cm × 1.2 cm) were made using the Au-coated silicon wafer as the starting substrate. They were spin-rinsed-dried using ST 270D (Semitool, CA) for a total of 400 s before spin-coating the polyimide to form a 4-µm-thick layer. Cross-linking involved exposure to UV light at 350 nm for 12 s and curing at 150 °C for 30 min and at 250 °C for an additional 30 min. This process completely covers the Au macrochip with polyimide so that the metal does not influence subsequent surface-modification experiments. The Au-coated and polyimide-coated silicon wafers were diced to size by hand using a diamond scribe. Self-Assembled Monolayers. The Au macrochips were cleaned in piranha solution (30:70 (v/v) of 30% H2O2 and concentrated H2SO4) for 30 min and thoroughly rinsed for 30 min with running DI water before use. (Caution: piranha solution is strongly oxidizing and should be handled with care!) SAM preparation, rinsing, and drying were carried out completely in an Ar-purged glovebag after the cleaning step to

eliminate oxidation of SAMs by air (or ozone).28-33 The cleaned Au macrochips were soaked in solutions of 4 mM MUA in Arpurged ethanol for 24 h to form SAMs, followed by rinsing with Ar-purged ethanol three times in each of three separate test tubes inside the glovebag. The chips were dried with Ar and kept in Ar-filled closed vials before use. Immobilization of the Primary DNA Probe. A working solution of 50 µg/mL P1 (unless otherwise indicated) and 0.1 M EDC in PB was prepared by combining appropriate volumes of stock solutions of 1000 µg/mL P1 in 10 mM TE buffer and 0.2 M EDC in PB, followed by dilution with PB buffer. All of the following steps for P1 immobilization were performed inside a glovebag filled with Ar. MUA SAM-modified macrochips were soaked in 500 µL of the P1/EDC working solution inside a capped polypropylene centrifuge for 2 h. The EDC assists covalent attachment of P1 to the free end of the SAMs.34-37 The chips were rinsed three times with 2× SSC and then soaked three times in 1× SSC for ∼15 min each.38 Capture of Synthetic Target, the DNA Gene for mRNA of hsp 70 for C. parvum, and Cross-Reactivity Tests with Targets of Other Organisms. Working solutions of 50 µg/mL T (unless otherwise indicated) were prepared by dilution of a 1000 µg/mL T in 10 mM TE buffer with 20× SSC/BSA. The P1immobilized macrochips were exposed to a 500-µL solution of 50 µg/mL T for 1 h inside a Parafilm-sealed Petri plate to prevent evaporation. The P1+T immobilized macrochips were soaked and shaken three times with 1 mL of 2× SSC for 30 min followed by 1 mL of 1× SSC for 15 min. Completing the DNA-Hybridization Assay Assembly with P2-AP. A working solution of 50 µg/mL P2-AP (unless otherwise indicated) was prepared by diluting a 1000 µg/mL P2-AP stock solution in 10 mM TE buffer with 20× SSC. P1-immobilized macrochips that had been exposed to T and rinsed were subsequently exposed to 500 µL of the P2-AP working solution for 3 h and then rinsed by soaking three times in 5 mL of acetate TBSA for 15 min each to eliminate nonspecifically adsorbed P2AP.39-41 These steps were performed outside of the glovebag, but inside Parafilm-sealed Petri plates to prevent evaporation. Enzymatic Generation of PAPR. The enzyme substrate solution was 4 mM PAPP in 0.1 M Tris at pH 9.0, as previously (28) Schoenfish, M. H.; Pemberton, J. E. J. Am. Chem. Soc. 1998, 120, 45024513. (29) Cooper, E.; Legget, G. J. Langmuir 1998, 14, 4795-4801. (30) Scott, J. R.; Baker, L. S.; Everett, W. R.; Wilkins, C. L.; Fritsch, I. Anal. Chem. 1997, 69, 2636-2639. (31) Zhang, Y.; Terrill, R. H.; Tanzer, T. A.; Bohn, P. W. J. Am. Chem. Soc. 1998, 120, 2654-2655. (32) Norrod, K. L.; Rowlen, K. L. J. Am. Chem. Soc. 1998, 120, 2656-2657. (33) Hutt, D. A.; Legget, G. J. J. Phys. Chem. 1996, 100, 6657-6662. (34) Gooding, J.; Hibbert, D. B. Trends Anal. Chem. 1999, 18, 525-533. (35) Zull, J. E.; Reed-Mundell, J.; Lee, Y. W.; Vezenov, D.; Ziats, N. P.; Anderson, J. M.; Sukenik, C. N. J. Ind. Microbiol. 1994, 13, 137-143. (36) Rasmussen, S. R.; Larsen, M. R. R., S. E. Anal. Biochem. 1991, 198, 138142. (37) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317-2323. (38) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. (39) Butler, J. E. Immunochemistry of Solid-Phase Immunoassay, 1st ed.; CRC Press: Boca Raton, FL, 1991. (40) Niwa, O.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1993, 65, 15591563. (41) Turner, A. P. F.; Newman, J. D. In Biosensors for Food Analysis; Scott, A. O., Ed.; The Royal Society of Chemistry: Cambridge, U.K., 1998.

described.23,42-47 The PAPP solution was purged with Ar and kept from light to avoid oxidation.42,44-47 Macrochips, containing the complete hybridization assembly, were rinsed three times with 5 mL of 0.1 M Tris at pH 9.0 at 10 min each before soaking in 5 mL of Ar-purged (15-30 min) PAPP solution for 12 h in a sealed beaker wrapped in aluminum foil inside a glovebag filled with Ar. The 12-h incubation time was chosen arbitrarily and was sufficient to achieve a high enough concentration of enzymatically produced PAPR in the large 5-mL volume to yield in an easily detectable signal. Control experiments verified that conversion of PAPP to PAPR over this 12-h period under these conditions does not occur when alkaline phosphatase is absent. Although we optimized the immobilization chemistry of the assay (i.e., the ratios of P1 and P2), we did not optimize the incubation time of PAPP on the macroscopic scale. This is because ultimately we would like to transfer the assay to the microscale. While the immobilization chemistry should not be affected significantly by downsizing, the incubation times for PAPP will (e.g., 30 s, as in the microelectrochemical immunoassay for mouse IgG).48 Surface Characterization. Surface composition at the various stages of surface modification on Au macrochips was studied using polarization-modulation Fourier transform infrared reflectance absorption spectroscopy (PM-FT-IR) with a Mattson Instruments Research Series 1 instrument. The IR beam was focused onto the sample at an incident angle of 77°. The reflected p-polarized beam was passed through a ZnSe Series II photoelastic modulator (Hinds) operating at 37 kHz before reaching the cooled HgCdTe detector. Spectra were taken with 4-cm-1 resolution and a halfwavenumber of 2900 cm-1. PM-FT-IR spectra were normalized by fitting the differential reflectance spectrum of the various stages of the surface-modification process between 4000 and 2100 cm-1 and between 2500 and 800 cm-1 to third-order polynomial backgrounds using FitIT curve-fitting software (Mattson). After curve fitting, the spectra were truncated and converted to absorbance using a WinFirst macro, written in-house under the specifications of Mattson. The sample chamber was purged with dry CO2-free air from a Balston air-dryer (Balston, Inc., Haverhill, MA). Each modified chip was kept in a scintillation vial (VWR) filled with Ar prior to PM-FT-IR analysis. Electrochemical Measurements. A BAS-100B potentiostat fitted with a PA-1 preamplifier (Bioanalytical Systems, Lafayette, IN) or a CH Instruments electrochemical workstation model 650A potentiostat with a picoampere booster and Faraday cage (CH Instruments, Inc, Austin, TX), controlled by a PC were used to perform cyclic voltammetry (CV). CV is a useful technique for diagnosing electrochemical behavior, especially in the initial stages of developing a DNA-hybridization assay. Although CV is not (42) Foulds, N. C.; Frew, J. E.; Green, M. J. In Biosensors A Practical Approach; Cass, A. E., Ed.; IRL Press, Oxford University: Oxford, U.K., 1990; pp 97124. (43) Bauer, C. G.; Eremenko, A. V.; Ehrentreich-Forster, E.; Bier, F. F.; Makower, A.; Halsall, H. B.; Heineman, W. R.; Scheller, F. W. Anal. Chem. 1996, 68, 2453-2458. (44) Xu, Y.; Halsall, H. B.; Heineman, W. R. J. Pharm. Biomed. Anal. 1989, 7, 1301-1311. (45) Duan, C.; Meyerhoff, M. E. Anal. Chem. 1994, 66, 1369-1377. (46) Thompson, R. Q.; Porter, M.; Stuver, C.; Halsall, H. B.; Heineman, W. R.; Buckley, E.; Smyth, M. R. Anal. Chim. Acta 1993, 271, 223-229. (47) Tang, H. T.; Lunte, C. E.; Halsall, H. B.; Heineman, W. R. Anal. Chim. Acta 1988, 214, 187-195. (48) Aguilar, Z. P.; Fakunle, E. S.; Fritsch, I. In preparation.

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usually the method of choice for quantitative electrochemistry, it was used for quantitation here so that we could compare it to CV responses reported in previous work.48 The electrochemical setup (Figure 1) involved Au macrochip working electrode, Pt flag auxiliary electrode, and Ag/AgCl (saturated KCl) reference electrode in a 25-mL screw cap beaker. Initial electrochemical characterization of working electrodes was performed in a solution containing 4 mM K3Fe(CN)6 in 0.1 M KCl and 4 mM PAPR or PAPP in 0.1 M Tris, pH 9.0. Activity of the enzyme-linked DNA-hybridization assay at macrochips of Au (and at macrochips of bare and double-sided polyimide-coated silicon wafers for nonspecific adsorption studies) was determined by evaluating the surrounding PAPP solution for PAPR. Working electrode potentials were kept within appropriate ranges to avoid electrochemical conversion of PAPP into PAPR.44 The Au underlying the modifying layer containing assay components was never used to detect the enzymatically generated PAPR. Tests for Cross-Reactivity of hsp70 mRNA from HeatShocked Whole Organisms. The vendor for the C. parvum oocysts, C. muris oocysts, and G. lamblia cysts heat shocked the microorganisms in a water bath for 10 min at 43 °C prior to shipping. The heat-shocking produces hsp mRNA, kills the microorganism, and ruptures some of them (microscopic analysis by vendor after heat shocking showed 7% excystation or rupture of the oocysts), releasing mRNA into the surrounding solution. It is the released mRNA that we can analyze. The mRNA is assumed to be fairly stable in the solution at this point, because RNAdegrading enzymes are presumably inactivated by the heating and the microorganisms are dead. We did not work with the live parasites ourselves, because our laboratory does not meet biohazard class III laboratory safety requirements. The samples from the vendor were diluted in our laboratory to 2 × 103 oocyst/mL from the (0.26 ( 0.05) × 106 oocysts/mL stock solution with 10 mM TE buffer in a 1.5-mL centrifuge tube. The solutions containing 2 × 103 oocyst/mL were heat shocked again in an attempt to release additional mRNA inside intact microorganisms by placing the tube in a beaker of water at 43-45 °C for 10 min. The mRNA in the solution was analyzed using the same procedure as that for the C. parvum target DNA, using P1 and P2-AP at 50 µg/mL. E. coli and S. aureus, both heat shocked and not heat shocked were evaluated. E. coli and S. aureus were prepared in the laboratory by streaking on agar plates and incubating at 37 °C for 24 h. A single colony of each organism was scraped and dissolved in 1 mL of 10 mM TE buffer in a 1.5-mL polyethylene centrifuge tube and used as a source for the target for the DNAhybridization assay. A duplicate of the solution of each organism was heat shocked in a beaker of water at 43-45 °C for 20 min. The resulting heat-shocked solution was also used as a source for the target following the procedure for the DNA-hybridization assay for C. parvum, using P1 and P2-AP at 50 µg/mL. To demonstrate that heat shocking releases mRNA, a colony of E. coli dissolved in 1 mL of TE buffer was heat shocked in a beaker of water at 43-45 °C for 20 min and treated with 1 mL Tri Reagent to isolate the RNA. A 400-µL drop of chloroform was subsequently added. The solution was vortexed for 15 s and allowed to stand at room temperature for 15 min before centrifugation at 10000g for 10 min. The aqueous phase was pipetted out 3894

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into a new tube and treated with 0.5 mL of 2-propanol. The solution was stored at room temperature for 5 min and then centrifuged at 10000g for 8 min. The RNA was a gellike pellet that settled at the bottom of the tube. To the RNA pellet was added 1 mL of 75% ethanol, followed by centrifugation at 7500g for 8 min. The ethanol was removed, and the pellet was air-dried at room temperature. The RNA was dissolved in 1 mL of FORMazol and incubated for 10 min at 55-60 °C before the absorbances at 260 and 280 nm were measured using a diode array spectrophotometer (HewlettPackard, 8452A). The RNA preparation gave an absorbance reading at both wavelengths between 1.6 and 1.8, which is positive for RNA.49 Optimization of P1 and P2-AP Concentrations. To evaluate the effect of P1 coverage, the assay was carried out using chips modified with P1 from solutions having concentrations of 5-75 µg/mL. Fixed concentrations of T and P2-AP at 50 µg/mL were used in these studies. To evaluate the effect of P2-AP, assays at concentrations of 5-75 µg/mL (based on the mass of the nonconjugated P2) were prepared with fixed concentrations of T and P1 at 25 µg/mL. RESULTS AND DISCUSSION Infrared Characterization of Modified Gold Macrochips. MUA SAMs as a platform for DNA immobilization have been previously reported. 50,51 However, to our knowledge, this is the first report of using MUA SAMs for immobilizing a DNA probe to gold surfaces in an enzyme-linked DNA hybridization assay for detecting C. parvum hsp70 mRNA. PM-FT-IR spectra of Au macrochips at the various stages of surface modification were obtained (see Supporting Information). At the low-wavenumber regions, peaks were observed for CdO ν at ∼1750 cm-1 from the purine and at 1680 cm-1 from the pyrimidine bases,52 PO2- ν asymmetric at 1220 cm-1 and symmetric at 1080 cm-1,52 and NH2 and N-H δ at 850 cm-1, which are typical of DNA.53-55 Bands in the amide region (CdO ν amide I at 1700 cm-1, N-H ν amide II at 1500 cm-1, and C-N ν amide III at 1400 cm-1) only appear after immobilization of P2-AP, as expected due to the presence of the protein AP. Activity of the Complete and Incomplete Assemblies of the Assay Components on Gold Macrochips for Synthetic DNA Target. The activity of modified surfaces was investigated by soaking macrochips coated with gold on one side in a solution of 4 mM PAPP in 0.1 M Tris for 12 h and detecting the enzymatically generated PAPR amperometrically at a nearby bare gold working electrode with Pt auxiliary and Ag/AgCl (saturated KCl) reference electrodes as in Figure 1. The target used in these studies was the synthetic DNA gene for hsp70 mRNA. CV responses for experiments where the macrochips were modified with the complete assay assembly and for those where at least one component was omitted were obtained (see Supporting (49) Molecular Research Center, Inc., 2002. (50) Schouten, S.; Stroeve, P.; Longo, M. L. Langmuir 1999, 15, 8133-8139. (51) Zhao, Y.; Pang, D.; Hu, S.; Wang, Z.; Cheng, J.; Dai, H. Talanta 1999, 49, 751-756. (52) Miller, L. M.; Carr, G. L.; Jackson, M.; Dumas, P.; Williams, G. P. Synchroton Radiat. News 2000, 13, 31-37. (53) Rao, C. N. Chemical Applications of Infrared Spectroscopy; Academic Press: New York, 1963. (54) Pretsch, E.; Simon, W.; Seibl, J.; Clerc, T. Tables of spetral data for structure determinations of organic compounds, 2nd ed.; Springer-Verlag: Berlin, 1989. (55) Conley, R. T. Infrared Spectroscopy; Allyn and Bacon, Inc.: Boston, 1966.

Figure 2. Nonspecific adsorption of active components of the assay on various materials. CV responses (50 mV/s) to enzymatically generated PAPR were obtained in 4 mM PAPP in 0.1 M Tris buffer after a 12-h incubation of macrochips that had been carried through the steps required for complete assembly (MUA SAM + EDC-coupled P1 + T + P2-AP). The macrochip was an oxidized silicon wafer with the following coatings: none (thin solid curve), both sides with gold (thick solid curve), one side with gold (medium solid curve), and both sides with polyimide (dotted curve).

Information). The largest peak current appeared when the complete assembly was involved. Small peak currents (10-25% of that for the complete assembly) were measured for incomplete assemblies. It was hypothesized that this small signal may be attributed to nonspecific adsorption on the silicon dioxide side of the macrochips. Investigation of Nonspecific Adsorption. Studies were performed to identify the extent of nonspecific adsorption on silicon dioxide, polyimide, and gold. Minimizing nonspecific adsorption is important to avoid false positives and high background results in an assay. Figure 2 shows a signal for PAPR, produced from a double-sided gold macrochip that was modified with the complete assembly (T ) synthetic DNA). The peak current is less than twice that from a silicon wafer coated only on one side with gold. This may be explained by the presence of a small degree of nonspecific adsorption on the silicon wafer. This explanation is consistent with the small signal measured when a bare silicon wafer chip was used. The polyimide macrochip gave no significant signal, indicating absence of nonspecific adsorption on that material. This result is promising for future work with devices that are made from polyimide. Tests for Cross-Reactivity. Environmental, drinking water, and fecal samples may contain mRNA from other microorganisms that can interfere with the DNA-hybridization assay for hsp70 mRNA of C. parvum. Synthetic DNA for hsp70 mRNA of C. parvum and for other naturally occurring pollution indicators and pathogenic organisms56-58 and samples of heat-shocked and nonheat-shocked cells of whole organisms were assayed to determine interferences. The synthetic DNA is expected to bind to P1 and P2 as well as the mRNA for which it codes.18 The purified target (56) University, W. Total Coliform Bacteria, http://wilkes.edu/∼eqc/coliform. htm, Center for Environmental Quality, September 24, 2001. (57) Todar, K. Todar’s Online Textbook of Bacteriology, http://www.textbookofbacteriology.net/, Kenneth Todar, May 29, 2002. (58) Ivnitski, D.; Abdel-Hamid, I.; Atanasov, P.; Wilkins, E. Biosens. Bioelectron. 1999, 14, 599-624.

Figure 3. Cross-reactivity tests of assay (complete assembly) using double-sided gold macrochips and oligonucleotides as the target from hsp 70 mRNA gene of various organisms. CV was performed in a solution of 4 mM PAPP in 0.1 M Tris at 50 mV/s that was incubated with the modified macrochip for 12 h. The responses correspond to the following target organisms: C. parvum (thick solid curve), and almost complete overlap for C. lari (dashed curve), E. coli (dotted curve), G. lamblia (medium solid curve), S. typhi (short dashed curve), and L. monocytogenes (thin solid curve). Concentrations: P1 ) 50 µg/mL, T ) 50 µg/mL, and P2-AP ) 50 µg/mL.

allows assessment of the assay for the oligonucleotide without complications from other cellular components. The section of synthetic DNA from other organisms was chosen based on the largest number of overlapping sequences with P1 and P2. Figure 3 compares results for the synthetic hsp70 mRNA from C. parvum with oligonucleotide sequences from E. coli, C. lari, G. lamblia, Li. monocytogenes, and S. typhi. There is no significant signal for enzymatically generated PAPR from any of the other targets. Therefore, the DNA-hybridization for hsp70 mRNA is highly specific to the oligonucleotide sequence for C. parvum. Samples of heat-shocked whole organisms allow evaluation of the extent of interferences from cellular components. The organisms were heat shocked twice to ensure rupture of cellular membranes that lead to emergence of the mRNA analyte into the surrounding fluid to make it accessible for detection. The organisms chosen for heat shock are based on previously reported studies13 and on organisms that were easily available to our laboratory, all of which are possible interferences from real samples. Figure 4 compares the electrochemical signal for the enzymatically generated PAPR from assays of heat-shocked whole organisms: C. parvum oocysts, C. muris oocysts, G. lamblia cysts, E. coli cells, and S. aureus cells. Reactivity to heat-shocked organisms other than C. parvum (at conditions that were found to show a positive signal for the presence of total RNA from heat-shocked E.coli) is negligible. These results suggest that the assay holds great promise for assays of real samples where such interferences may be present. Optimization of Concentrations Used in the Assay. Using synthetic oligonucleotides for the target hsp 70 mRNA of C. parvum, concentrations of P1 and P2-AP were varied during modification of macrochips to determine conditions that maximize the electrochemical signal. Figure 5 shows peak current density for CV responses from assays where macrochips were modified with concentrations of P1 ranging from 0.45 to 6.75 µM (5-75 µg/mL) and where concentrations of T and P2-AP were held constant at 1.8 (25 µg/mL) and 3.8 µM (50 µg/mL) (based on mass of unconjugated P2), respectively. Analytical Chemistry, Vol. 75, No. 15, August 1, 2003

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Figure 4. Cross-reactivity tests of the assay using solutions of heatshocked microorganisms. CV was performed in a solution of 4 mM PAPP in 0.1 M Tris at 50 mV/s that was incubated with the modified macrochip for 12 h. The responses correspond to the following target organisms: C. parvum at 2 × 103 oocyst/mL (thick solid curve), G. lamblia at 2 × 103 cysts/mL (dotted curve), C. muris at 2 × 103 oocyst/ mL (thin solid curve), E. coli heat shocked and not heat shocked at single 24-h colony/mL (complete overlap-dashed curve), and S. aureus heat shocked and not heat shocked at single 24-h colony/mL (complete overlap-short dash curve).

Figure 5. Optimization of probe (P1 and P2) concentrations to maximize the electrochemical signal. The concentration of P1 for coupling to the MUA SAMs was varied from 5 to 75 µg/mL (open circles), while T and P2-AP were held at fixed concentrations (25 µg of P1/mL and 50 µg of P2/mL, respectively). The concentration of P2AP used for the final step of the assembly was varied from 5 to 75 µg/mL (solid triangles), where the mass is based on the nonconjugated P2, while P1 and T were held at fixed concentrations (25 µg/ mL each). Current values were measured from CV responses obtained at 50 mV/s.

The electrochemical signal increases linearly with P1 concentration but plateaus when the concentrations of P1 and T are similar. This one-to-one correspondence between P1 and T suggests a quantitative immobilization of P1 and T from solution. As the P1 concentration in solution increases, the surface coverage on the modified macrochip also increases, as long as the maximum coverage is not reached. When the number of P1 molecules on the surface exceeds the number of T molecules in the subsequent solution, all available T molecules immobilize, achieving a complete surface coverage. Thus, they hybridize to a constant number of P2-AP molecules, resulting in a plateau in the electrochemical current. 3896 Analytical Chemistry, Vol. 75, No. 15, August 1, 2003

Figure 5 also demonstrates assay results when the concentration ofP2-AP is varied between 0.38 (5 µg/mL) and 5.7 µM (75 µg/mL) while T and P1 are maintained at constant concentrations of 1.8 and 1.9 µM (25 µg/mL), respectively. The electrochemical signal increases linearly with P2-AP concentration but plateaus when the concentration reaches that of the T solution. These data further support the hypothesis that the oligonucleotides in the solutions over the macrochip are quantitatively immobilized onto the surface in a one-to-one ratio and in a form that is active. An important conclusion is that, under these conditions, the assay should involve concentrations for P1 and P2-AP that are higher than that of T so that the signal is proportional to the T concentration and not limited by the probe concentrations. Such complete hybridization from solution should be much more rapidly achieved (within seconds or minutes) from smaller solution volumes using miniaturized devices (microliters to picoliters), such as the microcavity device that we previously reported.2 For example a ∼2-3-nL drop of solution on one of the microcavities was quantitatively electrolyzed in less than 5 min.26 Transfer to small devices such as this would greatly improve the incubation times and response times for detection. Sensitivity and Detection Limit. To evaluate the sensitivity and detection limit of the assay, a calibration curve over concentrations of T from 5 to 50 µg/mL was obtained (see Supporting Information). Fixed concentrations of P1 and P2-AP were selected (50 µg/mL) so that they would not limit the signal. The peak current from CV responses (average of two measurements) is linear, giving a least-squares fit of y ) (0.77 µA mL cm-2 µg-1)x - 0.97 µA cm-2, R2 ) 0.99. A limit of detection of 2 µg/mL (or 146 nM) was calculated at the 99% confidence level (t is ∼3 and there are >8 degrees of freedom), where the slope from the calibration curve is 0.77 ( 0.02 µA mL cm-2 µg-1, and the standard deviation from the blank signal (10 measurements) from 5 mL of 4 mM PAPP is 0.56 µA cm-2. This limit of detection is lower than previously reported by Wang and co-workers at 50 µg/mL.22 Furthermore, not only does our approach possess the capability to distinguish viable from nonviable oocysts, but it is also better suited for electrochemical detection than that described by Wang et al.22 because our detecting electrode is independent of the modified surface, giving a strong signal without perturbing the modified surface. Prospects for Miniaturization, Associated Detection Limits, and Time of Assay. By comparing the measured peak current from enzymatically generated PAPR from a 4 mM PAPP solution to that from a 4 mM PAPR solution (4000 µA/cm2), we can calculate that for the synthetic DNA only 1% of the PAPP was converted by the immobilized AP. This indicates that there is much opportunity to improve detection limits if the volume-tomodified surface area ratio is decreased significantly in order to concentrate the species, as would be possible in a miniaturized system (e1 µL volumes), such as the microcavity system that we previously reported for immunoassay of mouse IgG.2 In addition, if detecting electrodes are constructed immediately adjacent to the modified surface, as in our previous microcavity devices, detectable signals for enzymatically generated PAPR could be generated in seconds. With the previous device, it took less than 30 min to both complete the assembly of immunoassay components onto the antibody-modified surface and detect enzymatically

generated species. By downsizing, in a similar way, the DNAhybridization and electrochemical detection scheme for C. parvum described here, we expect that the assay time should decrease to less than 1 h. We also estimate that a limit of detection of 0.05 oocysts/mL (or 0.2 nM for the synthetic oligonucleotide) for hsp70 mRNA using the microcavity approach should be possible. If these detection limits were achieved, the microelectrochemical assay would be competitive with an assay reported by Esch et al.19 for hsp70 mRNA of C. parvum involving a microfluidic chip. They used off-chip nucleic acid sequence-based amplification, immobilization to gold within the microfluidic channel, and fluorescence-filled liposome labels to obtain a lowest detectable amount of 0.4 nM starting synthetic target oligonucleotide in 12.5 µL of amplified sample. Transfer of our electrochemical assay to detect hsp70 mRNA from live C. parvum oocysts in miniaturized systems, such as those we previously used for detection of mouse IgG,2 is planned. Certainly, if this approach is combined with procedures to release additional mRNA from intact oocysts or to amplify mRNA, the detection limits would be further improved. CONCLUSIONS To our knowledge, the enzyme-linked DNA-hybridization assay with electrochemical detection is a new technique for analysis of C. parvum that offers improvements over existing EPA detection methods and the possibility for distinguishing between viable and nonviable oocysts.5,8-13 Using oocysts subjected to heat shock, the assay time from capture of the oocysts to the detection of the electrochemical signal with the macrochip assay takes less than 20 h to complete. It eliminates the time-consuming steps involved in the immunofluorescence detection of the EPA methods such as dye staining, organism fixation, and microscopic quantitative analysis without necessarily eliminating the existing EPA filtration and immunomagnetic separation method. Normal assay time with the existing EPA method could take 4 days at least from the day the sample has been filtered and the oocysts isolated by immunomagnetic separation. In addition, electrochemical detection does not require extensive human involvement in the identification and

quantification steps of the immunofluorescence microscopy in the EPA methods. It should be possible to achieve proper heating conditions to amplify the mRNA as well. Extended heating can then be used to kill the oocysts and release the mRNA for detection. Specificity for C. parvum over other organisms using the assay is extremely high and is another advantage over the antibody-based EPA methods. Dramatic improvements to size, time of assay, and detection limits are anticipated for our electrochemical approach when this method is miniaturized (such as we did for detection of IgG)2 where modified surfaces and detecting surfaces are separate, yet located close together and within a confined volume. Actual improvements that can be made by downsizing must be demonstrated by future experiments. ACKNOWLEDGMENT We are grateful for the support of this research through grants from the National Science Foundation (NSF) (CHE-0096780) and from the Center for Sensing Technology and Research (CSTAR, funded by NSF, EPS-9977778, and the Arkansas Science and Technology Authority, 00-ARMF-06). We acknowledge additional support to Z.P.A. through an American Chemical Society Division of Analytical Chemistry Graduate Fellowship (Summer 2002) funded by Johnson and Johnson Pharmaceutical Research and Development. We also thank Mr. James E. Hoelscher, Jr., of Beaver Water District (Lowell, AR) for his insightful discussions about water pathogens and the needs of drinking water utilities. SUPPORTING INFORMATION AVAILABLE PM-FT-IR spectra of Au macrochips at various stages of modification, CV in PAPP solutions in which macrochips of complete and incomplete assemblies were incubated, and a calibration curve for T.. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review October 8, 2002. Accepted May 16, 2003. AC026211Z

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