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In this paper, a method of electrokinetic concentrating and a glass nanofluidic device for sensitivity-enhancing of magnetic beads-based antigen---antibody immunoassay (MBAAI) are presented. In the nanofluidic device, a polyacrylamide gel plug is deployed as the nanopores via photopolymerization reaction. To verify the capability of electrokinetic concentrating, the fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) with initial concentration of 0.002 ng/ml is enriched and the concentration factor of a hundred million fold is achieved. And then the technique is implemented to improve the MBAAI sensitivity. In this experiment, magnetic beads coupled with sheep anti-rabbit IgG (immunoglobulin G) are firstly trapped into the immunoassay region via magnetic force, then the FITC-labeled rabbit IgG is electrokinetically concentrated and spontaneously bonded with the previous fixed magnetic beads. After the incubation, the total fluorescence intensity in immunoassay region is calculated. The result shows that the fluorescence intensity is improved by 58 % and the antigen-concentrating with the nanofluidic device can effectively enhance the sensitivity of MBAAI.

Glutamic-pyruvic transaminase (GPT) is one of the most important enzymes in human liver and has a valuable clinical reference for the diagnosis of many liver diseases. Here a method for the determination of GPT activity based on transdeamination is presented. In this method, a three electrode setup was used for the cyclic voltammetric determination of the enzyme. With the electrochemical detection of reduced nicotinamide adenine dinucleotide (NADH) produced from transdeamination, the GPT activity was characterized under optimal circumstances. Firstly, to verify the response of the electrode to NADH, a series of NADH concentrations varying from 39 μM to 2.5 mM were calibrated with cyclic voltammetry (CV). A linear relationship between the NADH concentration and the peak current with R2 0.9999 was obtained. Then the concentration of α-ketoglutarate (α-KG) which can exert great influence on transdeamination was also optimized and the most sensitive response was achieved at the point of 0.75 mM α-KG. Finally, the GPT activity was determined using both the unmodified screen-printed carbon electrode (SPCE) and the electrode modified with CNTs. The results showed that the relationship between the GPT activity and the peak current of the CV curve was linear between 60 U L−1 and 300 U L−1 and the modified electrode exhibited a slightly better linear relationship than the unmodified electrode. This work proposes a new enzymatic reactive system based on transdeamination for the electrochemical detection of GPT activity and combines the electrochemical detection of NADH with the determination of GPT activity.

The activity of glutamic pyruvic transaminase (GPT) is an important clinical evidence for some acute diseases such as acute hepatopathy and myocardial infarction. Thus, there is a demand for rapid determination of GPT in small formats at point-of-need. Herein, we describe a novel method of electrochemical determination of GPT with microfluidic technique. GPT activity was indirectly determined via the electrochemical (EC) detection of nicotinamide adenine dinucleotide (NADH) produced from the GPT transdeamination reaction. A type of microfluidic chip was developed, in which a passive mixer comprising 100 sub-ribs and a three-electrode strip for EC were integrated. To verify the response to NADH, a series of NADH concentrations varying from 19 µM to 5 mM were calibrated with cyclic voltammetry within the microfluidic chip. And a linear relationship with R 2 0.9982 between the peak current and the concentration of NADH was obtained. Then, the GPT activity was determined using the chips containing and not containing a ribs-type mixer. And a linear relationship which contained two sections between the GPT activity and the peak current was obtained. The chip with a ribs-type mixer exhibited the sensitivity of 0.0341 μA U−1 L in the range of 10–50 U L−1 and 0.0236 μA U−1 L in the range of 50–250 U L−1. And the detection limit of the chip with a ribs-type mixer was 9.25 U L−1. The complete detection process of GPT activity within the microfluidic chip was realized, and the time-consuming problem was remarkably improved too.