Warakorn Limbut

Highly-sensitive label-free electrochemical carcinoembryonic antigen immunosensor based on a novel Au nanoparticles-graphene-chitosan nanocomposite cryogel electrode.

For the first time, a simple and highly sensitive label-free electrochemical carcinoembryonic antigen (CEA) immunosensor based on a cryogel electrode has been developed and tested. The as-prepared nanocomposite combined the advantages of the graphene, AuNPs and chitosan (AuNPs-GP-CS) together with the ease of preparing a cryogel coupled to a silver deposition, to act as a redox mediator, on a Au electrode. Under the optimal conditions, the decrease of the cyclic voltammetry (CV) silver peak current was proportional to the CEA concentration over a range of from 1.0×10(-6) to 1.0 ng mL(-1) with a detection limit of 2.0×10(-7) ng mL(-1). This AuNPs-GP-CS cryogel electrode gave a 1.7 times higher sensitivity and 25 times lower detection limit than the non-cryogel electrode. Moreover, the proposed electrochemical immunosensor exhibited good selectivity, reproducibility and stability. When applied to analyse clinical serum samples, the data determined by the developed immunosensor were in agreement with those obtained by the current hospital analysis system (enzyme linked fluorescent assay) (P>0.05), to indicate that the immunosensor would be potentially useful for clinical diagnostics.

Label-free capacitive immunosensors for ultra-trace detection based on the increase of immobilized antibodies on silver nanoparticles

Detection of ultra-trace amounts of antigens by label-free capacitive immunosensors was investigated using electrodes modified with silver nanoparticles (AgNPs) that allows for an increase in the amount of immobilized antibodies. The optimal amount of AgNPs that provided the highest immobilization yield was 48 pmol (in 2.0 mL). The performances of immunosensor electrodes for human serum albumin prepared with AgNPs, were compared to electrodes prepared with gold nanoparticles. The two systems provided the same linear range (1.0×10(-18) to 1.0×10(-10) M) and detection limit (1.0×10(-18) M). The system with AgNPs was used to analyze albumin in urine samples and the results agreed well with the immunoturbidimetric assay (P>0.05). Electrodes modified with AgNPs and appropriate antibodies were tested for their performances to detect analytes of different sizes. For a macromolecule (human serum albumin) the incorporation of AgNPs improved the detection limit from 100 to 1 aM. For small molecules, microcystin-LR and penicillin G, the detection limits were lowered from 100 and 10 fM to 10 and 0.7 fM, respectively. The high sensitivity and very low detection limits are potentially useful for the analysis of toxins or residues present in samples at ultra-trace levels and this method could easily be applied to other affinity pairs.

Label-free capacitive DNA sensor using immobilized pyrrolidinyl PNA probe: effect of the length and terminating head group of the blocking thiols.

This paper reports, for the first time, the influence of the length and the terminating head group of blocking thiols on the sensitivity and specificity of a label-free capacitive DNA detection system using immobilized pyrrolidinyl peptide nucleic acid (acpcPNA) probes. A C-terminal lysine-modified acpcPNA was immobilized through four different alkanethiol self-assembled monolayers (SAMs), i.e., 3-mercaptopropionic acid (MPA), thioctic acid (TA), thiourea (TU) and mercaptosuccinic acid (MSA). The hybridization between the acpcPNA probes and the target DNA was directly measured using the capacitive system. Five blocking thiols of various lengths (C=3, 6, 8, 9 and 11), with the -OH terminating head group, i.e., 3-mercapto-1-propanol (3-MPL), 6-mercapto-1-hexanol (6-MHL), 8-mercapto-1-octanol (8-MOL), 9-mercapto-1-nonanol (9-MNL), 11-mercapto-1-undecanol (11-MUL) and another blocking thiol (C=11) with a -CH(3) terminating head group, and 1-dodecanethiol (1-DDT) were investigated. The blocking thiol with the same length as the total spacer of the immobilized acpcPNA gave the highest sensitivity and specificity with the -OH terminating head group providing a slightly better signal than the -CH(3) group. Under the optimized conditions, the immobilized acpcPNA probes provided a wide linear range for DNA detection (1.0 × 10(-11)-1.0 × 10(-8)M) with a very low detection limit in the picomolar range. The modified acpcPNA electrode could be reused through at least 58 cycles. The high sensitivity and very low detection limits are potentially useful for the analysis of ultra-trace levels of DNA in samples. Preliminary studies were also performed to see the effect of probe concentration and target length.

Affinity sensor using 3-aminophenylboronic acid for bacteria detection

Boronic acid that can reversibly bind to diols was used to detect bacteria through its affinity binding reaction with diol-groups on bacterial cell walls. 3-aminophenylboronic acid (3-APBA) was immobilized on a gold electrode via a self-assembled monolayer. The change in capacitance of the sensing surface caused by the binding between 3-APBA and bacteria in a flow system was detected by a potentiostatic step method. Under optimal conditions the linear range of 1.5×10(2)-1.5×10(6) CFU ml(-1) and the detection limit of 1.0×10(2) CFU ml(-1) was obtained. The sensing surface can be regenerated and reused up to 58 times. The method was used for the analysis of bacteria in several types of water, i.e., bottled, well, tap, reservoir and wastewater. Compared with the standard plate count method, the results were within one standard deviation of each other. The proposed method can save both time and cost of analysis. The electrode modified with 3-APBA would also be applicable to the detection of other cis-diol-containing analytes. The concept could be extended to other chemoselective ligands, offering less expensive and more robust affinity sensors for a wide range of compounds.

4-mercaptophenylboronic acid functionalized gold nanoparticles for colorimetric sialic acid detection

A simple and selective colorimetric sensor for sialic acid detection, based on the aggregation of 4-mercaptophenylboronic acid functionalized gold nanoparticles (4-MPBA-AuNPs) was developed. The color of the solution changed from wine-red to blue after binding with sialic acid. The colorimetric sensor provided good analytical performances with a linear dynamic range of 80µM to 2.00mM and a 68±2µM limit of detection without any effect from possible interferences and sample matrix. In addition, the quantitative results were obtained within only 10min. This developed sensor was used to detect sialic acid in blood serum samples and the results were in good agreement with those from the current periodate-resorcinol method (P>0.05) thus indicating that this developed colorimetric sensor can be used as an alternative method for sialic acid detection with a shorter analysis time and a high accuracy.

Development and application of a real-time capacitive sensor

A real-time capacitive sensor based on a potentiostatic step method was developed. It can display in real-time the evoked current waveform, capacitance and the electrical resistance of elements serially connected to the insulation layer on the electrode as a function of time as well as the ohmic resistance of the insulation layer. These features enable the user to observe the association and dissociation of the affinity binding pairs and to evaluate the insulating property of the electrode surface during measurement. The system allows the setting of potential pulse height, pulse interval, gain, filter, and sampling frequency, enabling the system to be more flexible. The performance of the system was firstly evaluated with equivalent circuits. Under suitable parameter settings it provided good accuracy of both the capacitance and resistance. Using the affinity binding pair of human serum albumin (HSA) and anti human serum albumin (anti-HSA) the measured capacitance change was used for the direct detection of HSA. The developed system provided the same sensitivity as the commercially available potentiostat (P>0.05). The proposed system was then applied to analyse HSA in real urine samples and the results agreed well with the immunoturbidimetric assay (P>0.05). The proposed system can be applied for capacitance measurement to directly detect other target analytes using different affinity binding pairs. Other applications such as kinetics analysis of the interaction between affinity bindings, thickness analysis, and the study of the insulation property of the modified layer are also promising.

Ultra trace analysis of small molecule by label-free impedimetric immunosensor using multilayer modified electrode.

A multilayer electrode modified with a self-assembled thiourea monolayer (SATUM) followed by gold nanoparticles (AuNPs), mercaptosuccinic acid (MSA) and antibody was investigated for the detection of ultra trace amount of a small molecule (chloramphenicol) in an impedimetric system. The formation of the antibody-antigen complex at the electrode surface caused the impedance to increase. Under optimum conditions three modified electrodes were compared the SATUM/AuNPs/MSA electrode provided a wide linear range (0.50-10) × 10⁻¹⁶ M, and a very low determination limit of 1.0 × 10⁻¹⁶ M. This determination limit was much lower than the SATUM/AuNPs electrode, 1.0 × 10⁻¹⁵ M, and SATUM electrode, 4.7 × 10⁻¹⁴ M. The modified electrode provided good selectivity for chloramphenicol detection and can be reused up to 45 times with a relative standard deviation of lower than 4%. When applied to determine chloramphenicol in shrimp samples, the results agreed well with those obtained by the high-performance liquid chromatography coupled with a photo diode array detector (P > 0.05). The developed system can be applied to detect other small molecules using appropriate affinity binding pairs.

Electrochemical detection of the disease marker human chitinase-3-like protein 1 by matching antibody-modified gold electrodes as label-free immunosensors

Tissue inflammation, certain cardiovascular syndromes and the occurrence of some solid tumors are correlated with raised serum concentrations of human chitinase-3-like protein 1 (YKL-40), a mammalian chitinase-like glycoprotein, which has become the subject of current research. Here we report the construction and characterization of an electrochemical platform for label-free immunosensing of YKL-40. Details of the synthesis of YKL-40 and production of anti-YKL-40 immunoglobulin G (IgG) are provided and cross-reactivity tests presented. Polyclonal anti-YKL-40 IgG was immobilized on gold electrodes and the resulting immunosensors were operated in an electrochemical flow system with capacitive signal generation. The strategy offered a wide linear detection range (0.1μg/L to 1mg/L) with correlation coefficients (R(2)) above 0.99 and good sensitivity (12.28±0.27nF/cm(2) per decade of concentration change). Additionally, the detection limit of 0.07±0.01μg/L was well below that of optical enzyme-linked immunosorbent assays (ELISAs), which makes the proposed methodology a promising alternative for YKL-40 related disease studies.

Development of low-cost microfluidic systems for lab-on-a-chip biosensor applications

In this work, we develop low-cost microfluidic systems based on polydimethylsiloxane (PDMS) for lab-on-a-chip applications. PDMS microfluidic structures have been fabricated by micromolding, PDMS casting, and plasma bonding processes. The micromolding technique is used to fabricate PDMS slabs with micro-sized grooves, and the complete microchannel is formed by bonding PDMS slab with glass or PDMS substrate. The molding procedure using SU-8 photoresist patterning on silicon wafer, PDMS microchannel fabrication, and PDMS surface treatment using oxygen plasma and TiO2 coating, are discussed. The various parameters for oxygen plasma treatment including RF power and treatment time are studied in order to obtain conditions for good bonding with the glass substrate. The best condition for plasma treatment is found to be the low RF power (8 W) with 5 min treatment time. In addition, TiO2 coating with oxygen plasma treatment has been applied to make PDMS surface more hydrophilic to improve aqueous solution compatilbility. The microfluidic channels for various applications, including sample injection cross channel, micropump channel, T and Y sample mixers, PCR thermocyling chamber and channel, capillary electrophoresis flow channel, and conductimetric systems have been fabricated. Finally, a typical application of the PDMS chip in a flow injection conductimetric system for sodium chloride detection has been demonstrated.

Pyrrolidinyl PNA polypyrrole/silver nanofoam electrode as a novel label-free electrochemical miRNA-21 biosensor

A label-free electrochemical miRNA biosensor was developed based on a pyrrolidinyl peptide nucleic acid (acpcPNA)/polypyrrole (PPy)/silver nanofoam (AgNF) modified electrode. The AgNF was electrodeposited as redox indicator on a gold electrode, which was then functionalized with an electropolymerized layer of PPy, a conducting polymer, to immobilize the PNA probes. The fabrication process was investigated by electrochemical impedance spectroscopy. The biosensor was used to detect miRNA-21, a biomarker abnormally expressed in most cancers. The signal was monitored by the change in current of the AgNF redox reaction before and after hybridization using cyclic voltammetry. Two PNA probe lengths were investigated and the longer probe exhibited a better performance. Nucleotide overhangs on the electrode side affected the signal more than overhangs on the solution side due to the greater insulation of the sensing surface. Under optimal conditions, the electrochemical signal was proportional to miRNA-21 concentrations between 0.20fM and 1.0nM, with a very low detection limit of 0.20fM. The biosensor showed a high specificity which could discriminate between complementary, single-, doubled-base mismatched, and non-complementary targets. Three out of the seven tested plasma samples provided detectable concentrations (63 ± 4, 111 ± 4 and 164 ± 7fM). The sensor also showed good recoveries (81-119%). The results indicated the possibilities of this biosensor for analysis without RNA extraction and/or amplification, making the sensor potentially useful for both the prognosis and diagnosis of cancer in clinical application.