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A sensitive and rapid sandwich immunoassay (IA) was developed for human lipocalin-2 (LCN2) by functionalizing a KOH-treated polystyrene microtiter plate with multiwalled carbon nanotubes (MWCNTs) dispersed in 3-aminoproyltriethoxysilane (APTES). The significantly increased surface area due to the presence of MWCNTs led to a high immobilization density of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) activated anti-LCN2 capture antibodies (Ab). The anti-LCN2 Ab-bound MTPs were stable for 6 weeks when stored in 0.1 M PBS, pH 7.4 at 4 °C. The IA detects LCN2 from 0.6 to 5120 pg mL −1 with a limit of detection (LOD) and limit of quantification (LOQ) of 0.9 pg mL −1 and 6 pg mL −1 , respectively. The assay offered a ~50-fold lower LOD and ~3-fold faster IA, compared to a commercial sandwich enzyme-linked immunosorbent assay kit. [ABSTRACT FROM AUTHOR]

A surface plasmon resonance (SPR) biosensor has been developed for rapid immunoassay of procalcitonin (PCT) with high detection sensitivity and reproducibility. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-activated protein A (PrA), diluted in 1% (v/v) 3-aminopropyltriethoxysilane (APTES) was dispensed on a KOH-treated Au-coated SPR chip, resulting in the covalent binding of PrA in 30 min. This “single-step” PrA immobilization strategy led to the oriented binding of the anti-PCT antibody (Ab) on a PrA-functionalized gold (Au) chip. The leach-proof immobilization procedure is five-fold faster than conventional counterparts, enabling high detection specificity and reproducibility. The IA detects 4–324 ng mL −1 of PCT with a limit of detection (LOD) and a limit of quantification (LOQ) of 4.2 ng mL −1 and 9.2 ng mL −1 , respectively. It was capable of detecting PCT in real sample matrices and patient samples with high precision. The Ab-bound SPR chips were stable for more than five weeks. [ABSTRACT FROM AUTHOR]

A rapid sandwich enzyme-linked immunosorbent assay (ELISA)-based in vitro diagnostic (IVD) procedure has been developed for human fetuin A (HFA), an important disease biomarker for inflammatory diseases as well as malignancies. In this simplified and cost-effective procedure, the EDC-activated anti-HFA antibody (Ab) was admixed with 1% (v/v) 3-aminopropyltriethoxysilane (APTES) in 1:1 (v/v) and dispensed in a KOH-pretreated microtiter plate (MTP). APTES formed a stable complex with the capture antibody that was in turn covalently bonded on the KOH-treated surface in 30 min. The resulting immunoassay (IA) format detects HFA with a dynamic range of 0.1–243 ng mL −1 , and a limit of detection (LOD) and analytical sensitivity of 0.3 ng mL −1 and 1.0 ng mL −1 , respectively. For the determination of HFA spiked in diluted human whole blood and serum, and HFA in ethylenediaminetetraacetic acid (EDTA)-plasma of patients, the obtained analytical precision is similar to that of the conventional sandwich ELISA. The anti-HFA Ab-bound MTPs, stored at 4 °C in 0.1 M PBS, pH 7.4, retained its biological activity for 8 weeks, thereby demonstrating excellent storage stability. This generic sandwich ELISA procedure can be extended for rapid, simplified and cost-effective detection of other disease biomarkers. [ABSTRACT FROM AUTHOR]

Abstract: We have developed a highly sensitive immunoassay using graphene nano platelets (GNPs) for the rapid detection of human lipocalin-2 (LCN2) in plasma, serum, and whole blood. It has the dynamic range, linear range, limit of detection, and analytical sensitivity of 0.6 to 5120, 80 to 2560, 0.7, and 1pg/ml, respectively. It is the most sensitive assay for the detection of LCN2, which has 80-fold higher analytical sensitivity and 3-fold lesser immunoassay duration than the commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit. The functionalization of microtiter plate (MTP) with GNPs, dispersed in 3-aminopropyltriethoxysilane (APTES), provided the increased surface area that leads to higher immobilization density of capture antibodies. Moreover, the generation of free amino groups on MTP and GNPs by APTES enables the leach-proof covalent crosslinking of anti-human LCN2 capture antibody by its carboxyl groups using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as the heterobifunctional crosslinker. The anti-LCN2 antibody-bound MTPs were highly stable given that they did not show any significant decrease in their functional activity when stored at 4°C in 0.1M phosphate-buffered saline (PBS) for 8weeks. The developed immunoassay correlated well with the conventional ELISA, thereby demonstrating its high precision and potential utility for highly sensitive analyte detection in industrial and clinical settings. [Copyright &y& Elsevier]

Abstract: A mediatorless glucose biosensor was developed by the immobilization of glucose oxidase (GOx) to graphene-functionalized glassy carbon electrode (GCE). The surface of GCE was functionalized with graphene by incubating it with graphene dispersed in 3-aminopropyltriethoxysilane (APTES), which acted both as a dispersion agent for graphene and as an amine surface modification agent for GCE and graphene. This was followed by the covalent binding of GOx to graphene-functionalized GCE using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) based crosslinking. Graphene provided signal enhancement by providing greater surface area for GOx binding, while APTES-functionalization led to a higher GOx immobilization density by providing free amino groups for crosslinking. The developed biosensor used a redox potential of −0.45V (vs. Ag/AgCl) for detecting glucose in the diabetic pathophysiological range 0.5–32mM. There was no interference from endogenous electroactive substances and drug metabolites. The developed biosensor was further validated for detecting blood glucose in commercial artificial blood glucose linearity standards in the range 1.4–27.9mM. Therefore, it is ideal for diabetic blood glucose monitoring. The developed bioanalytical procedure for preparation of GOx-bound graphene-functionalized GCEs had high production reproducibility and high storage stability, which is appropriate for the commercial mass production of enzyme-bound electrodes. [Copyright &y& Elsevier]

Abstract: Chemiluminescent enzyme immunoassay (CLEIA), surface plasmon resonance (SPR) immunoassay and enzyme- linked immunosorbent assay (ELISA) were developed for the highly sensitive detection of human albumin (HA). The bioanalytical procedure, involving the surface modification and antibody immobilization, was the same for all immunoassay formats. The bioanalytical platforms, i.e. microtiter plates (MTP) and SPR gold chips, were initially functionalized with 3-aminopropyltriethoxysilane and then crosslinked to anti-HA antibodies using 1-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide. The developed HA immunoassay formats were compared on the basis of their analytical performance. CLEIA was found to be the best format for HA detection as it had the highest analytical sensitivity with lowest limit of detection and widest dynamic range. The analytical sensitivity of various immunoassay formats were in the decreasing order of CLEIA>ELISA>SPR. The developed CLEIA for HA detection was 6-fold more sensitive than the widely used commercially-available ELISA. The anti-HA antibody bound MTPs, stored at 4°C in 0.1M PBS, pH 7.4, were stable for up to 4 weeks, and can be effectively used for the rapid detection of HA in just 2.5h. [Copyright &y& Elsevier]

Abstract: A rapid sandwich enzyme linked immunoassay (ELIA) procedure was developed for the highly sensitive detection of human Lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL). It reduced the total immunoassay duration by more than 3-fold and detected LCN2 in the pathophysiological range of 2.5-5120 pg/mL with an analytical sensitivity of 7 pg/mL, which is about 11-fold better than that of the commercially-available conventional enzyme linked immunosorbent assay (ELISA). The enhanced analytical performance of developed ELIA procedure is due to the leach-proof covalent crosslinking of anti-human LCN2 antibody to the 3- aminopropyltriethoxysilane (APTES)-functionalized microtiter plate (MTP) by 1-ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride. The multisubstrate-compatible ELIA procedure enables LCN2 assay on different types of polymeric substrates (inert, hydrophilic and hydrophobic) using the modified MTP format. The developed ELIA procedure can be similarly employed to develop rapid and highly sensitive immunoassays for other disease biomarkers and analytes in clinical and industrial settings. [Copyright &y& Elsevier]

Abstract: We developed a method to completely regenerate the gold (Au) surface of 3-aminopropyltriethoxysilane (APTES)-functionalized Au-coated surface plasmon resonance (SPR) chip that had been used for human fetuin A (HFA) immunoassay. It involved treatment of the used SPR chip with freshly prepared piranha solution (concentrated H2SO4/30% H2O2 =3:1, v/v) for 15min followed by extensive rinsing with ethanol and deionized water. The developed method enabled prolonged reuse of the regenerated SPR chip that increased its cost-effectiveness without affecting the reproducibility of HFA immunoassays. [Copyright &y& Elsevier]