Modified Gold Electrode Surface Research Articles

Development of label-free electrochemical OMP-DNA probe biosensor as a highly sensitive system to detect of citrus huanglongbing

The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)6]3−/4− on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease.

Potential Induced Protonation of the Second Coordination Sphere in a Hangman-type Iron Porphyrin Complex Promotes HER: Insights via in Situ Raman Spectroelectrochemistry.

The iron-based porphyrin complex containing a bispyridine-based hanging unit termed Py2XPFe was previously used as an effective catalyst for the reduction of protons to molecular hydrogen in solution. Here, the molecular compound was immobilized on a modified gold electrode surface and investigated by spectroelectrochemical methods under catalytic conditions. Immobilization of the Py2XPFe was facilitated using a pyridine-based amine linker molecule grafted to the gold electrode by electrochemical amine oxidation. The linker molecule denoted in this report as Pyr-1 allows for effective coordination of the iron porphyrin compound to the modified gold surface through axial coordination of the pyridine component to the Fe center. Resonance Raman spectroelectrochemistry was performed on the immobilized catalyst in pH 7 buffer at increasing cathodic potentials. This facilitates the electrochemical hydrogen evolution reaction (HER) while concurrently allowing for the observation of the v4, v3, and v2 porphyrin marker bands, which are sensitive to oxidation and spin state changes at the metal center. The observed changes in these bands at decreasing potential indicate that the immobilized Py2XPFe exists in the formal high-spin FeIII state before being reduced to the low-spin FeII state resulting from axial interaction with the linker moiety. This FeII state likely acts as the precatalyst for the HER reaction. Surfaced enhanced Raman spectroelectrochemistry was also conducted on the system as the gold electrode provides a sufficient surface enhancement effect so as to observe the bonding nature of the pyridine substituents within the second coordination sphere. As the potential is lowered cathodically, the pyridine ring breathing modes at 999 cm-1 are shown to increase in intensity due to protonation, which reach an intensity saturated limit whereat HER is conducted. This suggests that in pH 7 buffer, the increase in cathodic potentials facilitates protonation of the pyridine-based second coordination sphere. The extent to which protonation occurs can be viewed as a function of decreasing potential due to an increase in proton flux at the immobilized catalyst which, at the required onset potential for catalysis, aids in the reduction of protons to molecular hydrogen.

Methylthiophenyl- and Methylthiobiphenyl-Substituted A2B CoIIIcorroles: Modulating Electrocatalyzed Hydrogen Evolution Reactions on Surface-Modified Gold Electrodes

Four A2B-type CoIIIcorroles (2a-2d) with electron-donating/withdrawing substituents at the A2 meso-aryl substituents and a 4-(methylthio)phenyl ring at the B position have been synthesized and characterized, along with a series of meso-extended CoIIIcorroles (4a-4c) with 4'-(methylthio)biphenyl moieties. The electronic structures and structure-property relationships of the dyes have been analyzed by comparing their redox and optical properties to trends predicted in density functional theory calculations. Au electrodes surface-modified with 2a-2d and 4a-4c are highly efficient catalysts for electrocatalyzed hydrogen evolution reactions, and the electrocatalytic properties can be readily modulated by fine-tuning the electronic structure of the CoIIIcorrole and the distance between the "Au-S" bond and CoIII center.

Expression, purification, characterization and direct electrochemistry of two HiPIPs from Acidithiobacillus caldus SM-1

High-potential iron-sulfur proteins (HiPIPs) from extremely acidophilic chemolithotrophic non-photosynthetic Acidithiobacillus commonly play a crucial role in ferrous or sulfurous biooxidation. Acidithiobacillus exhibit important industrial applications for bioleaching valuable metals from sulfide ores. In this study, two HiPIP genes from thermophilic Acidithiobacillus caldus SM-1 were cloned and successfully expressed, and their proteins were purified. The proteins displayed a brownish color with an optical absorbance peak at approximately 385 nm and an electronic paramagnetic resonance (EPR) g value of approximately 2.01, which confirmed that the iron-sulfur cluster was correctly inserted into the active site when the proteins were generated in E. coli. The proteins were more thermostable than HiPIPs from mesophilic Acidithiobacillus. The direct electron transfer (DET) between HiPIPs and electrode was achieved by the 2-mercaptopyrimidine (MP) surface-modified gold electrodes; the redox potentials of the HiPIP1 and HiPIP2 measured by cyclic voltammetry were approximately 304.5 mV and 400.5 mV, respectively. The electron transfer rate constant was estimated to be 0.75 s−1 and 0.66 s−1, respectively. The MP/Au electrode and Au electrode showed consistent differences in heterogeneous electron transfer rates and electron transfer resistances. Bioinformatics and molecular simulations further explained the direct electron transfer between the proteins and surface-modified electrode.

Surface functionalized gold electrode by axial ligand exchanged CoIIIcorroles for accelerating electrocatalyzed hydrogen evolutions

Herein, a series of CoIIIcorroles with different meso-substituents were successfully functionalized on gold electrodes through self-assembled axial coordination with 4-(methylthio)pyridine (MTP) and 4-(4-methylthiobenzo)pyridine (MTPP). The surface modified gold electrode performed enhanced and tunable electrochemically catalyzed hydrogen evolution behaviors that demonstrates the tunable surface molecular engineering of CoIIIcorroles via self-assembled monolayer is an effective strategy for energy related small molecule activations. Additionally, the CoIIIcorrole with electron-withdrawing meso-substituents axially coordinated with expanded conjugation system, 4-(4-methylthiobenzo)pyridine (MTPP), has the best performance of electrochemically catalyzed hydrogen evolutions.

Monolayer cobalt(II)phthalocyanine functionalized gold electrode for enhanced electrocatalyzed oxygen reductions

Monolayer Co(II)phthalocyanine (Co(II)Pc) functionalized gold electrodes through self-assembled axial coordination with 4-(methylthio)pyridine (MTP) and 4-(4-methylthiobenzo)pyridine (MTPP) were prepared. The surface modified gold electrode performed electrochemically catalyzed oxygen reduction in terms of onset-potential, current density, overpotential and Tafel in the order Au < Au/Co(II)Pc < Au/MTP/CoPc(II) < Au/MTPP/Co(II)Pc that demonstrates tunable surface molecular engineering of Co(II)Pc via self-assembled monolayer is an effective strategy for molecular catalysis and device application.

Electrochemical immunosensor for detection of Plasmodium vivax lactate dehydrogenase.

BACKGROUND Malaria is a disease that affects many tropical and subtropical countries, including Brazil. The use of tests for malaria detection is one of the fundamental strategies recommended by the World Health Organization for the control and eradication of the disease. The lack of diagnostic tests leads to an increase in transmission and non-reporting cases.OBJECTIVES This work described an electrochemical immunosensor for detecting Plasmodium vivax lactate dehydrogenase antigen (Ag-PvLDH).METHODS The device has developed by immobilising egg yolk IgY antibodies (Ab-PvLDH) on a gold electrode surface using cysteamine as linker. The immunosensor fabrication was followed by differential pulse voltammetry, and contact angle measurements were performed to characterise the modified gold electrode surface.FINDINGS The results for Ag-PvLDH determination exhibit a linear response at 10-50 µg mL-1 concentration range, with a limit of detection of 455 ng mL-1. The excellent selectivity of the device was confirmed.MAIN CONCLUSIONS The developed immunosensor showed a good performance, therefore, it can be considered an alternative test to detect malaria caused by P. vivax.

Electrochemical detection of DNA by formation of efficient electron transfer pathways through adsorbing gold nanoparticles to DNA modified electrodes

The high-performance detection of nucleic acids is increasingly important in the early and accurate disease diagnosis. Herein, we describe a unique label-free electrochemical DNA biosensor, which is based on gold nanoparticles (GNPs) mediated electron transfer through a DNA monolayer modified gold electrode surface. We observed that the different states of DNA monolayers (single-strand DNA or triple-helix DNA) and the different sizes of GNPs have a significant effect on the electron transfer by the DNA modified gold electrodes. This DNA biosensing strategy avoids the complicated fabrication process of the DNA capture probes and minimizes the steps of electrode pre-treatment. Under optimal conditions, this method can sensitively and accurately detect the target DNA ranging from 10 pM to 100 nM with a detection limit of 1.47 pM.

Ultrasensitive Electrochemical Biosensor Developed by Probe Lengthening for Detection of Genomic DNA in Human Serum.

As an alternative to most of the reported nucleic acid amplification-based electrochemical DNA biosensors used for detection of trace levels of genomic DNA, we herein present a novel detection concept. The proposed system involves the conversion of two short double-stranded DNAs (dsDNAs), labeled with a thiol-tag or biotin-tag, into a single integrated dsDNA containing thiol and biotin at both terminals in the presence of target DNA through ligase chain reaction (LCR) and followed by the immobilization of these integrated dsDNAs on a bovine serum albumin (BSA)-modified gold electrode surface. Owing to rapid depletion of the two short dsDNAs via LCR, the integrated dsDNAs were generated in an exponential manner so that this sensoring approach offered a limit of detection of 25 yoctomoles (15 copies in 50 μL sample volumes), a high discrimination of single-base mismatch and a wide linear concentration range (across 6 orders of magnitude) for target DNA. Significantly, the proposed sensor, which has simplicity in operation and ease of miniaturization, detected the target of interest in total nucleic acid extracts derived from clinical serum samples with excellent results, thereby demonstrating its considerable diagnostic potential in fields ranging from virus detection to the diagnosis of genetic diseases.

Electrocatalytic Behaviour of Surface Confined Pentanethio Cobalt (II)Binuclear Phthalocyanines Towards the Oxidation of 4-Chlorophenol

Cobalt binuclear phthalocyanine (CoBiPc) bearing pentanethio substituents at the peripheral positions were synthesized. The immobilization of the synthesized cobalt phthalocyanines on gold electrode was achieved using self-assembled monolayer method (SAM). X-ray photoelectron spectroscopy (XPS) and Kelvin Probe (KP) techniques were used to characterise the formation of monomeric and binuclear phthalocyanine SAMs on the gold surface. The phthalocyanine SAMs on gold electrodes were investigated for electrocatalytic oxidation of 4-chlorophenol. The electrocatalytic properties of tetra- and octa- pentanethio substituted cobalt binuclear phthalocyanine (CoBiPc) are compared with their tetra- and octa-pentanethio substituted phthalocyanine (CoPc). The SAMs modified gold electrode surfaces showed a peak current enhancement and stability and reduction in electrocatalytic potentials compared to the bare or unmodified electrodes towards the detection of the 4-chlorophenol. The SAMs of cobalt binuclear phthalocyanines exhibited more enhanced electrocatalytic properties in terms of stability, detection peak current and reduction of the electrocatalytic over potential reduction.

Electrocatalytic behaviour of surface confined pentanethio cobalt (II) binuclear phthalocyanines towards the oxidation of 4-chlorophenol

Cobalt binuclear phthalocyanine (CoBiPc) bearing pentanethio substituents at the peripheral positions were synthesized. The immobilization of the synthesized cobalt phthalocyanines on gold electrode was achieved using self-assembled monolayer method (SAM). X-ray photoelectron spectroscopy (XPS) and Kelvin Probe (KP) techniques were used to characterise the formation of monomeric and binuclear phthalocyanine SAMs on the gold surface. The phthalocyanine SAMs on gold electrodes were investigated for electrocatalytic oxidation of 4-chlorophenol. The electrocatalytic properties of tetra- and octa- pentanethio substituted cobalt binuclear phthalocyanine (CoBiPc) are compared with their tetra- and octa-pentanethio substituted phthalocyanine (CoPc). The SAMs modified gold electrode surfaces showed a peak current enhancement and stability and reduction in electrocatalytic potentials compared to the bare or unmodified electrodes towards the detection of the 4-chlorophenol. The SAMs of cobalt binuclear phthalocyanines exhibited more enhanced electrocatalytic properties in terms of stability, detection peak current and reduction of the electrocatalytic over potential.

A Novel Photo‐Induced Electrochemical Biosensing Method Based on Fluorescent Labeled Molecular Beacon

AbstractA novel photo‐induced electrochemical biosensing method has been developed based on fluorescence quenching effect and electrochemical method. In this sensing strategy, the molecular beacon probes labeled with methylene blue were immobilized on the gold nanoparticles modified gold electrode surface firstly; then dopamine was assembled on the electrode surface through electrostatic interaction with gold nanoparticles. Under the continuous illumination, the fluorescence of the methylene blue was quenched by the gold nanoparticles before hybridization; after hybridization with the complementary DNA, methylene blue was far away from the gold nanoparticles and the fluorescence recovered, and then singlet oxygen was generated in the photosensitive reaction of methylene blue in the presence of dissolved oxygen. Singlet oxygen reacted with dopamine, which resulted in the reduction of concentration of the dopamine on the electrode surface. The current of the dopamine on the electrode was used for the sensing of the conformational change of molecular beacon and hence for the detection of target DNA.

Oxime chemistry-mediated covalent capturing on electrode surface with guanidinium recognition and application for aldolase activity assay

A new strategy to fabricate electrochemical biosensor is proposed based on the covalent capturing of molecules on electrode interface through oxime chemistry and the loading of signal probes via guanidinium-phosphate electronic interaction with a “covalent-like” stability. Taking aldolase as an example, enzyme analysis can be achieved through the covalent loading of the catalyzed products on the aminooxy modified gold electrode surface and subsequent immobilization of guanidinium functionalized sliver nanoparticles. Different from adolase substrate, the hydrolyzed products contain both carbonyl groups and phosphate acid groups in their structures. The carbonyl groups can chemoselectively react with aminoxy groups on the electrode surface to form oxime, resulting in the exposure of phosphate groups. Subsequently, signal probe can be loaded onto electrode surface through guanidinium recognition, giving a electrochemical current value. So the number of enzyme catalyzed products determines the amount of signal probes on the electrode surface, and aldolase activity can be analyzed. For the proposed method, the chemoselective oxime ligation and highly stable guanidinium recognition improve the accuracy, selectivity and stability of enzyme analysis. The established method may be extended to the analysis of other related substances containing carbonyl groups.

PM-IRRAS Studies of DMPC Bilayers Supported on Au(111) Electrodes Modified with Hydrophilic Monolayers of Thioglucose.

A phospholipid bilayer composed of 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine (d54-DMPC) was deposited onto the Au(111) electrode modified with a self-assembled monolayer of 1-thio-β-d-glucose (β-Tg) via the Langmuir-Blodgett and Langmuir-Schaefer (LB-LS) techniques. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements were used to characterize structural and orientational changes in this model biological membrane on a hydrophilic surface modified gold electrode. The results of the spectroscopic measurements showed that the tilt angle of acyl chains obtained for deuterated DMPC bilayers supported on the β-Tg-modified gold is significantly lower than that reported previously for DMPC bilayers deposited directly on Au(111) electrodes. Moreover, tilt angles of ∼18° were obtained for d54-DMPC bilayers on β-Tg self-assembled monolayers (SAMs) at positive potentials, which are similar to the values calculated for h-DMPC deposited on bare gold in the desorbed state and to those observed for a stack of hydrated DMPC bilayers. This data confirms that the β-thioglucose SAM promotes the formation of a water cushion that separates the phospholipid bilayer from the metal surface. As a result, the DMPC polar heads are not in direct contact with the electrode and can adopt a zigzag configuration, which strengthens the chain-chain interactions and allows for an overall decrease in the tilt of the acyl chains. These novel supported model membranes may be especially useful in studies pertaining to the incorporation of peptides and proteins into phospholipid bilayers.

Gold electrode modified with ultrathin SnO2 nanosheets with high reactive exposed surface for electrochemical sensing of As(III)

Here we report a system using ultrathin SnO2 nanosheets with high reactive exposed (110) surface modified gold electrode for sensitive detection of As(III) by Square wave anodic stripping voltammetry (SWASV) with a mild acid condition (pH 5.0). Ultrathin SnO2 nanosheets have been synthesized via a hydrothermal process successfully with the thickness of about 0.52nm (four atomic layers thick). This oxide is used as electrode modifier, and the modified electrode exhibits good performance in stripping determination of As(III). The LOD actually measured is 5μgL−1 and below the guideline value given by the World Health Organization (WHO). As compared with SnO2 bulks, ultrathin SnO2 nanosheets are found to exhibit stronger adsorption capability and better detection performance toward As(III), it may be due to the atomic thickness and extremely huge percentage of (110) facet ultrathin nanosheets materials exposed. Finally, this system is successfully applied for the analysis of a real sample collected from Inner Mongolia, China. Most importantly, we pose a potential for the use of ultrathin materials to improve the electrochemical performance of bare gold electrode in mild acid condition.

Exploiting the Reaction of Cytochrome C with Fructose Dehydrogenase for a Multilayer Electrode Construction

The creation of analytical electron transfer (ET) chains based on the defined arrangement of enzymes and the redox protein cytochrome c (cyt c) on electrode surfaces represents an emerging field in bioelectronics.1,2 In this study we investigate the ET reaction of the flavin-dependent enzyme fructose dehydrogenase (FDH) with the redox protein cyt c. Two different pH optima are identified for the reaction with both proteins in solution, at acidic and neutral pH. When one reaction partner - cyt c - is immobilized on a modified gold electrode surface, ET proceeds efficiently at neutral pH which can be concluded from well shaped catalytic currents obtained in cyclic voltammograms. In addition a defined dependence on the substrate concentration is observed. In acidic media the reaction can also be verified but appears to be less efficient. This indicates that two different ET pathways between the enzyme and immobilized cyt c occur. Moreover, it is demonstrated that both partners can be assembled in a stable multilayer architecture, using the biopolymer DNA as connecting polyelectrolyte. The defined layered deposition of DNA with co-immobilized FDH and cyt c is verified by surface plasmon resonance (SPR) measurements. Prepared on electrodes, substantial catalytic currents are recorded upon addition of fructose. The response is found to be dependent on the number of layers deposited on the surface. This shows that an artificial signal chain can be constructed through multiple protein layers. Our results contribute to the better understanding of the ET-reaction between FDH and cyt c in a surface confined state, providing the basic knowledge which may be relevant for the creation of innovative fructose sensitive electrodes. 1. Lisdat, F.; Dronov, R.; Moehwald, H.; Scheller, F. W.; Kurth, D. G., Chemical Communications 2009, 274-283. 2. Feifel, S. C.; Kapp, A.; Lisdat, F., in Biosensors Based on Aptamers and Enzymes, Springer 2014, 140, 253-298.

Development of a very sensitive electrochemical immunosensor for the determination of 17β-estradiol in bovine serum samples

This study describes the development of a very sensitive electrochemical immunosensor (EI) for the determination of 17β-estradiol (17β-E). The novelty of this immunosensor is that the detection of 17β-E is carried out without sample pretreatment and unlabeled neither the antigen nor the antibody. Very good results in terms of sensitivity, kinetics, and working range are obtained. The immunosensor was constructed by immobilization of the anti-17β-E monoclonal antibody (mAbE) on a gold disk electrode modified with gold nanoparticles on a cysteamine self-assembled monolayer (AuNP-cys-Au disk). Bovine serum samples were spiked with known amounts of 17β-E and incubated on mAbE-AuNP-cys-Au disk electrodes. Then, the EI was transferred to pH 5.00 citrate buffer solutions, containing horseradish peroxidase (HRP), pyrocatechol (H2Q), and H2O2 at given concentrations. The 17β-E and H2Q, both enzyme co-substrates, react with HRP. The HRP, which did not react with 17β-E, in the presence of H2O2 catalyzes the oxidation of H2Q to o-benzoquinone (Q). The back electrochemical reduction of Q to H2Q was detected on the modified gold electrode surface by square wave voltammetry. The electrochemical signal was proportional to the amount of H2Q that reacts with the enzyme, and inversely proportional to the amount of 17β-E presents in the bovine serum samples. The EI showed a linear range from 0.54 to 1.36×104pgmL−1. The limit of detection (LOD) was 0.84pgmL−1. Recovery percentages were very good, with values of 99.7, 106, and 105% for 10, 50 and 100pgmL−1, respectively. This EI is an attractive tool for the 17β-E determination in bovine serum samples.

One-step, ultrasensitive, and electrochemical assay of microRNAs based on T7 exonuclease assisted cyclic enzymatic amplification.

Taking advantage of the special exodeoxyribonuclease activity of T7 exonuclease, a simple, sensitive, selective, and label-free microRNA biosensor based on the cyclic enzymatic amplification method (CEAM) has been proposed. First, thiol functionalized DNA probes were assembled onto a gold nanoparticles modified gold electrode surface through a Au-S bond, followed by hybridizing with target miRNA. Subsequently, DNA in RNA/DNA duplexes was digested by T7 exonuclease, which can release the microRNA molecules from the electrode surface and return into the buffer solution. Meanwhile, the released microRNA can further hybridize with the unhybridized DNA probes on the modified electrode surface. On the basis of it, an isothermal amplification cycle is realized. The T7 exonuclease-assisted CEAM achieved a low detection limit of 0.17 fM. Moreover, this assay presents excellent specificity with discriminating only a single-base mismatched microRNA sequence. Furthermore, this work can also be applied to detect avian leukemia based on the decreased expression level of microRNA-21.

Target-induced aptamer release strategy based on electrochemical detection of staphylococcal enterotoxin B using GNPs-ZrO2-Chits film

A novel electrochemical aptasensor was developed for ultrasensitive detection of staphylococcal enterotoxin B (SEB) by combining signal amplification and target-induced aptamer release strategy. A gold electrode was modified with a nanocomposite made of gold nanoparticles reduced in situ, zirconia nanoparticles, and chitosan. The SEB aptamer was hybridized by anchoring the capture probe on the modified gold electrode surface through AuS binding. In the presence of SEB, the capture probe-aptamer duplex was compelled to open, releasing the aptamer from the electrode. The resulting single-strand capture probe was hybridized with a biotinylated detection probe and labeled with streptavidin-horseradish peroxidase, producing an ultrasensitive enzyme-catalyzed electrochemical signal. Under optimal conditions, the amperometric responses were proportional to the SEB concentrations ranging from 2 to 512ngmL−1, with a detection limit of as low as 0.24ngmL−1 (S/N=3). The aptasensor exhibited good stability, outstanding reproducibility, and high selectivity. The as-prepared aptasensor was used to analyze SEB in human serum specimens, and validated through enzyme-linked immunosorbent assay method. Analytical results suggest that the developed assay is a promising alternative approach for detecting SEB in clinical diagnosis.

Construction of Biomimetic Interface by Layer-by-Layer Self-Assembly Technique for Electrochemical Amperometric Immunosensing Applications

Abstract A novel biomimetic interface for developing label-free amperometric immunosensor based on layer-by-layer (LBL) self-assembly of positively charged ferrocene-chitosan/multiwalled carbon nanotubes (CS-Fc/MWNTs) and negatively charged gold nanoparticles (Au NPs) onto 3-mercaptopropanesulfonic sodium salt (MPS)-modified gold electrode surface was described. Through LBL self-assembly of CS-Fc/MWNTs and Au NPs, the constructed three-dimensional mutilayer network structure could provide more steric sites and ideal platform for further immobilization of biomolecules without loss of their activity. By using hepatitis B surface antigen (HBsAg) as a model, the constructed immunosensor exhibited a specific response to HBsAg linearly in the concentration range of 0.1–350 ng mL−1 with a detection limit of 0.037 ng mL−1 (S/N = 3). The relative error between the results obtained from the immunosensor and the standard method proposed by ELISA was −4.46% to 8.33%, indicating that the two methods were in acceptable agreement and the proposed method could be applied to determination of HBsAg in human serum for clinical diagnosis.