Microfluidic chip-based electrochemical immunoassay for hippuric acid.

Urinary hippuric acid (HA), of molecular weight 180 Da, is one of the major metabolites in toluene-exposed humans and is a major biological indicator. Simple and ubiquitous monitoring of exposure to toluene is very important in occupational health care, and a microfluidic chip-based electrochemical immunoassay for rapid and quantitative detection of HA in human urine is proposed in this paper. The system employs a conjugate of ferrocene (Fc) and hippuric acid (HA). The competition between hippuric acid (HA) and the ferrocene-hippuric acid complex (Fc-Lys-HA) to bind with a HA antibody coated onto polybeads generated electrical signals proportional to the HA concentration in the range of 0-40 mg mL(-1). All the complicated HA detection processes were integrated on the single microfluidic platform. The quantitative advantages of our HA detection chip are as follows: (1) the total chip size was reduced to 3.0 x 2.0 x 0.5 cm and is small enough to be portable, (2) the assay time took 1 min, and is shorter than that of conventional electrochemical HA immunoassay systems (about 20 min) and (3) 40 microL of the sample solution was enough to detect HA in the range of 0-40 mg mL(-1), which is enough range to be used for the point-of-care system. In addition, we suggest the improved chip-based HA assay method by the combination of electrochemical and enzymatic amplification processes for the detection of greater electrical signals. The sensitivity of the combined method was increased about three times compared to that of the non-enzymatic process.

[1]  J. Khandurina,et al.  Bioanalysis in microfluidic devices. , 2002, Journal of chromatography. A.

[2]  T Kitamori,et al.  Integration of an immunosorbent assay system: analysis of secretory human immunoglobulin A on polystyrene beads in a microchip. , 2000, Analytical chemistry.

[3]  M. Meyerhoff,et al.  Separation-free sandwich enzyme immunoassays using microporous gold electrodes and self-assembled monolayer/immobilized capture antibodies. , 1994, Analytical chemistry.

[4]  T Kitamori,et al.  Determination of carcinoembryonic antigen in human sera by integrated bead-bed immunoassay in a microchip for cancer diagnosis. , 2001, Analytical chemistry.

[5]  Sang Hoon Lee,et al.  A pneumatically controllable flexible and polymeric microfluidic valve fabricated via in situ development , 2005 .

[6]  Thomas Laurell,et al.  Microfluidic enzyme immunoassay using silicon microchip with immobilized antibodies and chemiluminescence detection. , 2002, Analytical chemistry.

[7]  Jin-Woo Choi,et al.  A new magnetic bead-based, filterless bio-separator with planar electromagnet surfaces for integrated bio-detection systems , 2000 .

[8]  Richard S. Nicholson,et al.  Theory and Application of Cyclic Voltammetry for Measurement of Electrode Reaction Kinetics. , 1965 .

[9]  J Wang,et al.  Electrochemical enzyme immunoassays on microchip platforms. , 2001, Analytical chemistry.

[10]  G. Rivas,et al.  Glucose microsensor based on electrochemical deposition of iridium and glucose oxidase onto carbon fiber electrodes , 1997 .

[11]  K R Rogers,et al.  Thick-film electrochemical immunosensor based on stripping potentiometric detection of a metal ion label. , 1998, Analytical chemistry.

[12]  Ingrid Fritsch,et al.  Self-contained microelectrochemical immunoassay for small volumes using mouse IgG as a model system. , 2002, Analytical chemistry.

[13]  C. Filley,et al.  Central nervous system effects of chronic toluene abuse--clinical, brainstem evoked response and magnetic resonance imaging studies. , 1988, Neurotoxicology and teratology.

[14]  Guodong Liu,et al.  Quantum-dot-based electrochemical immunoassay for high-throughput screening of the prostate-specific antigen. , 2008, Small.

[15]  Jae Wook Lee,et al.  Electrochemical immunosensor using p-aminophenol redox cycling by hydrazine combined with a low background current. , 2007, Analytical chemistry.

[16]  Joseph Wang,et al.  Highly Selective Membrane-Free, Mediator-Free Glucose Biosensor , 1994 .

[17]  Rosanne M Guijt,et al.  Use of bioaffinity interactions in electrokinetically controlled assays on microfabricated devices , 2002, Electrophoresis.

[18]  D. J. Harrison,et al.  Microchip systems for immunoassay: an integrated immunoreactor with electrophoretic separation for serum theophylline determination. , 1998, Clinical chemistry.

[19]  H. S. Kim,et al.  Functionalization of a poly(amidoamine) dendrimer with ferrocenyls and its application to the construction of a reagentless enzyme electrode. , 2000, Analytical chemistry.

[20]  M. Okochi,et al.  Electrochemical killing of microorganisms using the oxidized form of ferrocenemonocarboxylic acid , 1999 .

[21]  Jagotamoy Das,et al.  A nanocatalyst-based assay for proteins: DNA-free ultrasensitive electrochemical detection using catalytic reduction of p-nitrophenol by gold-nanoparticle labels. , 2006, Journal of the American Chemical Society.

[22]  T. Matsunaga,et al.  Construction of electrochemical flow immunoassay system using capillary columns and ferrocene conjugated immunoglobulin G for detection of human chorionic gonadotrophin. , 2001, Biosensors & bioelectronics.

[23]  Tae-Kyu Lim,et al.  Microfabricated on-chip-type electrochemical flow immunoassay system for the detection of histamine released in whole blood samples. , 2003, Analytical chemistry.

[24]  M. Ogata,et al.  Direct colorimetric determination of hippuric acid in urine. , 1972, Clinical chemistry.

[25]  Ng Tp,et al.  Urinary levels of proteins and metabolites in workers exposed to toluene. A cross-sectional study. , 1990 .

[26]  H. Hurtig,et al.  Persistent cerebellar ataxia after exposure to toluene , 1977, Annals of neurology.

[27]  D. Erickson,et al.  Integrated microfluidic devices , 2004 .

[28]  S. Yanagihara,et al.  Simultaneous determination of hippuric acid and o-, m- and p-methylhippuric acids in urine by high-performance liquid chromatography. , 1983, Journal of chromatography.

[29]  G. S. Wilson,et al.  Flow injection immunoassays: A review , 1998 .

[30]  D. Kalman,et al.  Toluene metabolites as biological indicators of exposure. , 2002, Toxicology letters.

[31]  M. J. Joseph,et al.  Enzymatically amplified voltammetric sensor for microliter sample volumes of salicylate. , 1995, Analytical chemistry.

[32]  T. Ptak,et al.  Chronic toxic encephalopathy in a painter exposed to mixed solvents. , 1999, Environmental health perspectives.

[33]  R. W. Wright,et al.  Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. , 1986, Analytical biochemistry.

[34]  W. Heineman,et al.  Small volume bead assay for ovalbumin with electrochemical detection. , 2001, The Analyst.

[35]  P. Kongtip,et al.  Modified method for determination of hippuric acid and methylhippuric acid in urine by gas chromatography. , 2001, Journal of chromatography. B, Biomedical sciences and applications.

[36]  Amperometric Glucose Biosensor Based on Gold‐Dispersed Carbon Paste , 1998 .

[37]  T. Nakajima,et al.  Determination of benzene and toluene in blood by means of a syringe-equilibration method using a small amount of blood. , 1975, British journal of industrial medicine.

[38]  Adam Heller,et al.  Reduction of the nonspecific binding of a target antibody and of its enzyme-labeled detection probe enabling electrochemical immunoassay of an antibody through the 7 pg/ml-100 ng/mL (40 fM-400 pM) range. , 2005, Analytical chemistry.

[39]  Kyung Sun,et al.  Stable Deposition and Patterning of Metal Layers on the PDMS Substrate and Characterization for the Development of the Flexible and Implantable Micro Electrode , 2007 .

[40]  W. C. Purdy,et al.  Homogeneous Voltammetric Immunoassay: A Preliminary Study , 1979 .

[41]  Lapos,et al.  Injection of fluorescently labeled analytes into microfabricated chips using optically gated electrophoresis , 2000, Analytical chemistry.

[42]  C. Padeste,et al.  Ferrocene-avidin conjugates for bioelectrochemical applications. , 2000, Biosensors & bioelectronics.

[43]  J. Gergely,et al.  Zero-length crosslinking procedure with the use of active esters. , 1990, Analytical biochemistry.

[44]  T. Matsunaga,et al.  Miniaturized amperometric flow immunoassay system using a glass fiber membrane modified with anion. , 2002, Biotechnology and bioengineering.

[45]  Hyug-Han Kim,et al.  Immunochromatographic analysis of hippuric acid in urine. , 2007, Journal of analytical toxicology.