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An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Plasmodium falciparum has been developed. The target molecule was a segment of the repeat sequence of the gene coding for the circumsporozoite (CSP) protein from the AF54087 gene. This analyte offers the possibility of specifically detecting P. faleiparum. The assay involves attachment of a biotinylated primary DNA probe via its 5'-amineterminus to the streptavidin-coated surface of microwells in a 96-well plate. The primary DNA probe (1 [sup.0]P, which was of two different sequences we call 1[sup.0][P.sub.a] and 1 [sup.0][P.sub.b]) was used to capture the target (T, which was of two different sequences, [T.sub.1] sequence 481-590 and [T.sub.2] sequence 472-590 of AF54087 gene for the CSP gene) by hybridization to a complementary sequence on the target. On 1 [sup.0][P.sub.a], 47 bases were complementary to [T.sub.1] and [T.sub.2] at 543-590, while on 1 [sup.0][P.sub.b]], 35 bases were complementary to [T.sub.1] and [T.sub.2] at 555-590. A secondary DNA probe that contained 36 bases with alkaline phosphatase (2[sup.0]P-AP) label on the 3' end was hybridized to a complementary base sequence on the 5' end of the target, p-Aminophenol, which is enzymatically generated by the immobilized AP fromp-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammo-grams from a PAPP solution incubated inside the microwells modified with the complete assembly of the assay components gives a linear relationship with the concentration of the target (2-50 ng/mL, where [P.sub.1][ ([P.sub.1a] and [P.sub.1b]) and [P.sub.2]-AP concentrations are 50 ng/mL). A detection limit of 1.4 ng/mL (or 46 pM) of the DNA target was obtained. The signals of the assays were not significantly affected when performed in the presence of human hepatocytes, pig liver, or chicken serum indicating the viability of this assay in real clinical samples.

A new approach is reported for analysis of ultrasmall volumes. It takes advantage of the versatile positioning of a dispenser to shoot ~150-pL droplets of liquid onto a specific location of a substrate where analysis is performed rapidly, in a fraction of the time that it takes for the droplet to evaporate. In this report, the site where the liquid is dispensed carries out fast-scan cyclic voltammtry (FSCV), although the detection method does not need to be restricted to electrochemistry. The FSCV is preformed at a microactivity having individually addressable gold electrodes, where one serves as working electrode and another as counter/pseudoreference electrode. Five or six droplets of 10 mM [[Ru(N[H.sub.3]).sub.6]] [Cl.sub.3] in 0.1 M KCl were dispensed and allowed to dry, followed by redissolution of the redox species and electrolyte with one or five droplets of water and immediate FSCV, demonstrating the ability to easily concentrate a sample and the reproducibility of redissolution, respectively. Because this approach does not integrate detection with microfluidics on the same chip, it simplifies fabrication of devices for analysis of ultrasmall volumes. It may be useful for singlestep and multistep sample preparation, analyses, and bioassays in microarray formats if dispensing and changing of solutions are automated. However, care must be taken to avoid factors that affect the aim of the dispenser, such as drafts and clogging of the nozzle.

An electrochemical enzyme-linked immobilized DNA-hybridization 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 self-assembled monolayers of mercaptoundecanoic acid to a gold surface. The primary DNA probe was used to capture the target (sequence 1039-1082 of U71181 gene 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 (5-50 [micro]g/mL, where [P.sub.1] and [P.sub.2]-AP concentrations are 50 [micro]g/mL). A detection limit of 2 [micro]g/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.

A self-contained, microelectrochemical immunoassay on the smallest volumes reported to date (1 [micro]L for the antigen, 1 [micro]L for the secondary antibody-enzyme conjugate, and 200 nL for the electrochemically detected species) has been developed using mouse IgG as a model system in a sandwich-type enzyme-linked immunosorbant assay, which takes less than 30 min to both complete the assembly of immunoassay components onto the antibody-modified surface and detect enzymatically generated species (excluding time for electrochemical cleaning of electrodes). These studies demonstrate the advantage of the close proximity of electrodes to modified surfaces and their application in the analysis of small volumes. Using a 50 [micro]m diameter x 8 [micro]m deep cavity with individually addressable electrodes on a microfabricated chip, the primary antibody was selectively and covalently attached at a gold, recessed microdisk (RMD) at the bottom of the microcavity to the free end of SAMs of either 11-mercaptoundecanoic acid or 11-mercapto-1-undecanol using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. Nonspecific adsorption to the surrounding material, polyimide, of the microcavity device was eliminated. Electrochemical desorption was used to confine the immunoassay activity at the RMD. Enzymatic conversion of the substrate p-aminophenyl phosphate to p-aminophenol is detectable in less than 30 s using cyclic voltammetry at a gold, tubular nanoband electrode, which is on the wall of the microcavity and immediately adjacent to the modified RMD. A third electrode, also within the region of the microcavity, served as the pseudoreference/auxiliary electrode. Calibration curves obtained for 1-[micro]L solutions of 5-100 ng/mL of IgG and for 200 nL-solutions of 5 [micro]M to 4 mM of PA[P.sub.R] gave detection limits of 4.4 nM (6.4 ng/mL) or 880 fmol (129 pg) for PA[P.sub.R] and 56 fM (9 pg/ mL) or 56 zmol (9 fg) for IgG. It is expected that the device may be suitable for analysis with volumes down to tens of picoliters.

A Ru(phen)32+-doped silica fluorescent nanoparticle (FN)-based immunochromatographic test strip (ICTS) sensor was developed for rapid, high sensitivity, easy to use, and low cost quantitative detection of enrofloxacin (ENR) residues in chicken meat. The fluorescence signal intensity of the FNs at the test line (FIT) and control line (FIC) was determined with a prototype of a portable fluorescent strip reader. Unique properties of Ru(phen)32+ doped silica nanoparticles (e.g., large Stokes shift, high emission quantum yield, and long fluorescence lifetime) were combined with the advantages of ICTS and an easy to make portable fluorescent strip reader. The signal was based on FIT/FIC ratio to effectively eliminate strip to strip variation and matrix effects. Various parameters that influenced the strip were investigated and optimized. Quantitative ENR detection with the FNs ICTS sensor using 80 μL sample took only 20 min, which is faster than the commercial ELISA kit (that took 90 min). The linear range of detection in chicken extract was established at 0.025-3.500 ng/mL with a half maximal inhibitory concentration at 0.22 ± 0.02 ng/mL. Using the optimized parameters, the limit of detection (LOD) for ENR using the FNs ICTS sensor was recorded at 0.02 ng/mL in chicken extract. This corresponds to 0.12 μg/kg chicken meat which is two (2) orders of magnitude better that the maximum residue limits (MRLs) imposed in Japan (10 μg/kg) and three (3) orders of magnitude better than those imposed in China. The intra- and inter-assay coefficient of variations (CVs) were 6.04% and 12.96% at 0.5 ng/mL, 6.92% and 12.61% at 1.0 ng/mL, and 6.66% and 11.88% at 2.0 ng/mL in chicken extract, respectively. The recoveries using the new FNs ICTS sensor from fifty (50) ENR-spiked chicken samples showed a highly significant correlation (R² = 0.9693) with the commercial enzyme-linked immunosorbent assay (ELISA) kit. The new FNs ICTS sensor is a simple, rapid, sensitive, accurate, and inexpensive quantitative detection of ENR residues in chicken meat and extracts. [ABSTRACT FROM AUTHOR]

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Plasmodium falciparum has been developed. The target molecule was a segment of the repeat sequence of the gene coding for the circumsporozoite (CSP) protein from the AF54087 gene. This analyte offers the possibility of specifically detecting P. falciparum. The assay involves attachment of a biotinylated primary DNA probe via its 5'-amine-terminus to the streptavidin-coated surface of microwells in a 96-well plate. The primary DNA probe (1 0Pa, which was of two different sequences we call 1 0Pa and 1 0Pa) was used to capture the target (T, which was of two different sequences, T1 sequence 481-590 and T2 sequence 472- 590 of AF54087 gene for the CSP gene) by hybridization to a complementary sequence on the target. On 1 0Pa, 47 bases were complementary to T1 and T2 at 543-590, while on 1 0Pb, 35 bases were complementary to T1 and T2 at 555-590. A secondary DNA probe that contained 36 bases with alkaline phosphatase (2 0P-AP) label on the 3' end was hybridized to a complementary base sequence on the 5' end of 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 incubated inside the microwells modified with the complete assembly of the assay components gives a linear relationship with the concentration of the target (2-50 ng/mL, where P1 (P1a and P1b) and P2-AP concentrations are 50 ng/mL). A detection limit of 1.4 ng/mL (or 46 pM) of the DNA target was obtained. The signals of the assays were not significantly affected when performed in the presence of human hepatocytes, pig liver, or chicken serum indicating the viability of this assay in real clinical samples. [ABSTRACT FROM AUTHOR]

Presents an electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of cryptosporidium parvum that is capable of distinguishing dead from live oocysts and suitable for miniaturization and array formats. Capture of synthetic target, the DNA gene for mRNA of heat shock protein 70 for cryptosporidium parvum and cross reactivity tests with targets of other organisms; Characterization of surface at various stages of surface modification on Au macrochips using polarization; Infrared characterization of modified gold macrochips; Prospects for miniaturization, associated detection limits and time of assay.

Studies regarding circulating tumor cells (CTCs) have great significance for cancer prognosis, treatment monitoring, and metastasis diagnosis. However, due to their extremely low concentration in peripheral blood, isolation and enrichment of CTCs are the key steps for early detection. To this end, targeting the folic acid receptors (FRs) on the CTC surface for capture with folic acid (FA) using bovine serum albumin (BSA)-tether for multibiotin enhancement in combination with streptavidin-coated magnetic nanoparticles (MNPs-SA) was developed for ovarian cancer CTC isolation. The streptavidin-biotin-system-mediated two-step binding strategy was shown to capture CTCs from whole blood efficiently without the need for a pretreatment process. The optimized parameters for this system exhibited an average capture efficiency of 80%, which was 25% higher than that of FA-decorated magnetic nanoparticles based on the one-step CTC separation method. Moreover, the isolated cells remained highly viable and were cultured directly without detachment from the MNPs-SA-biotin-CTC complex. Furthermore, when the system was applied for the isolation and detection of CTCs in ovarian cancer patients' peripheral blood samples, it exhibited an 80% correlation with clinical diagnostic criteria. The results indicated that FA targeting, in combination with BSA-based multibiotin enhancement magnetic nanoparticle separation, is a promising tool for CTC enrichment and detection of early-stage ovarian cancer.

A Ru(phen)3(2+)-doped silica fluorescent nanoparticle (FN)-based immunochromatographic test strip (ICTS) sensor was developed for rapid, high sensitivity, easy to use, and low cost quantitative detection of enrofloxacin (ENR) residues in chicken meat. The fluorescence signal intensity of the FNs at the test line (FI(T)) and control line (FI(C)) was determined with a prototype of a portable fluorescent strip reader. Unique properties of Ru(phen)3(2+) doped silica nanoparticles (e.g., large Stokes shift, high emission quantum yield, and long fluorescence lifetime) were combined with the advantages of ICTS and an easy to make portable fluorescent strip reader. The signal was based on FI(T)/FI(C) ratio to effectively eliminate strip to strip variation and matrix effects. Various parameters that influenced the strip were investigated and optimized. Quantitative ENR detection with the FNs ICTS sensor using 80 μL sample took only 20 min, which is faster than the commercial ELISA kit (that took 90 min). The linear range of detection in chicken extract was established at 0.025-3.500 ng/mL with a half maximal inhibitory concentration at 0.22 ± 0.02 ng/mL. Using the optimized parameters, the limit of detection (LOD) for ENR using the FNs ICTS sensor was recorded at 0.02 ng/mL in chicken extract. This corresponds to 0.12 μg/kg chicken meat which is two (2) orders of magnitude better that the maximum residue limits (MRLs) imposed in Japan (10 μg/kg) and three (3) orders of magnitude better than those imposed in China. The intra- and inter-assay coefficient of variations (CVs) were 6.04% and 12.96% at 0.5 ng/mL, 6.92% and 12.61% at 1.0 ng/mL, and 6.66% and 11.88% at 2.0 ng/mL in chicken extract, respectively. The recoveries using the new FNs ICTS sensor from fifty (50) ENR-spiked chicken samples showed a highly significant correlation (R(2) = 0.9693) with the commercial enzyme-linked immunosorbent assay (ELISA) kit. The new FNs ICTS sensor is a simple, rapid, sensitive, accurate, and inexpensive quantitative detection of ENR residues in chicken meat and extracts.

Screen-printed gold (SPG, Dupont gold conductor 5734) on low-temperature co-fired ceramic (LTCC) materials (Dupont dielectric tape 951, mostly composed of alumina and silica) has been demonstrated to be a substrate for electrochemical enzyme-linked immunosorbant assays. The effect of two different cleaning treatments and the extent of nonspecific adsorption on the SPG/LTCC and plain LTCC surfaces were also evaluated. LTCC materials hold promise for constructing a new generation of devices for microelectrochemical sensing and assays. Facile fabrication in three dimensions with integrated conducting elements makes them attractive. A standard sandwich immunoassay for a model analyte, mouse IgG, was used to evaluate the LTCC materials. After the assembly of components onto chips of SPG/LTCC and plain LTCC, p-aminophenol that was generated enzymatically by the enzyme label was detected electrochemically with a separate glassy carbon electrode. Cleaning SPG/LTCC with a piranha solution (7:1 vol/vol of concentrated H2SO4/30% H2O2), traditionally used for other gold surfaces prior to SAM assembly, resulted in a notable decrease in assay signal and an increase in nonspecific adsorption when compared to cleaning with water alone. Assay components assemble specifically on plain LTCC, with only a small percent attributed to NSA. Environmental scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy reveal the tremendous chemical heterogeneity and complexity of both SPG/LTCC and plain LTCC surfaces and aid in the explanation of assay results. A 10% acetate Tween bovine serum albumin solution and an ethanolic solution of 4 mM 1-butanol eliminate assay signals originating from plain LTCC. The outcomes of these studies can now be used to achieve miniaturized electrochemical immunoassays on LTCC materials where both plain and SPG surfaces are present.