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Fluorescence capillary analysis (FCA) realizes trace-level analysis of micro-volume samples; it is easy to operate, extremely low in analytical cost and can significantly lessen environmental pollution from analytical chemistry waste. FCA has the characteristics of green analytical chemistry and has been applied in clinical, biochemical, pharmaceutical, food safety and other fields. FCA basically involves a micro-volume glass capillary, a capillary holder and an ordinary fluorescence detector. The capillary is not only a container for chemical reaction and detection but also functions as a carrier to immobilize enzymes, gene probes or reagents; it can be used repeatedly or can be disposable. In analysis, the capillary which is modified with functional reagents sucks in a measured liquid for the reaction and is then inserted into the holder within the fluorescent detector for measurement. The immobilized FCA method has been successfully used in the determination of reduced coenzyme I, ethanol in liqueur, lactic acid in dairy products, pyruvic acid and glucose in serum, trace-level sulfated bile acid in urine, the ratio of pyruvic/lactic acid in serum, and pyruvic acid in cells as well as in DNA end-labeling and dyeing methods. Further, FCA can also be extended to capillary arrays to complete multipurpose simultaneous determinations and can be combined with mobile phones as fluorescence detectors for use in mobile health analytical technology. FCA will produce considerable social benefits in medicine, pharmacy, fermentation of food, environmental protection and other fields. Therefore, the relevant contents are presented in this tutorial review.

Herein, a fluorescent capillary biosensor was developed for quantifying micro-volume intracellular pyruvate (PA), in which AuNPs and lactate dehydrogenase (LDH) were modified on the inner surface of an amination capillary (20 μL) via a self-assembly technique. The PA concentration was quantified by the change in the value of the fluorescence of NADH after sucking a mixed solution of the sample and NADH into the biosensor. This study investigated factors including the degree of protonation of the amino groups on the surface of the capillary, the AuNP concentration and time for self-assembly, the activity concentration and time for the LDH self-assembly, the flow rate and acidity for LDH immobilization, pH, temperature, and reaction time for the NADH/PA/LDH reaction system. Under the optimized conditions, the linear response range of the biosensor towards PA was 2.5-120 μmol L -1 , in which the determination limit and detection limit were 2.5 and 0.75 μmol L -1 , respectively. The biosensor could be reused more than 41 times when its relative standard deviation (RSD) was controlled at less than 1.5%. At room temperature (approximately 25 °C), the intracellular PA in the erythrocyte of a healthy person was measured using the biosensor, and the PA content was observed to be 241.76 ± 68.05 μmol L -1 (n = 8). The standard addition recovery was 95-106%. Employment of the AuNPs in the PA biosensor not only improved the affinity of the immobilized LDH towards PA and its stability, but also significantly enhanced the service life of the PA biosensor.

It was studied that making conditions of a micro-volume fluorescence capillary biosensor for determining pyruvate (PA) and lactate (LA). The biosensor made under the optimized conditions could be used for sequential quantifications of LA in the range 0.10-1.2 mM and PA in 4-120 μM, and its recovery for PA and LA was in a satisfactory range 97-106% for human serum samples, with detection limits of 0.023 mM for LA (RSD < 1.89%, n = 11) and 0.87 μM for PA (RSD < 1.70%, n = 11). The new assay possessed these advantages that the LDH immobilizing on capillary realized the reuse of expensive enzyme in fluorospectrophotometry, and the consumption of serum samples or chemical reagents decreased to 9 μL in per assay, and the analytes no needed to preseparation, and it also are accurate and reliable. Consequently, the fluorescence capillary biosensor should have a good prospect in assaying PA and LA or LA/PA ratios for clinical medicines or biology field. The optimization conditions and parameters obtained in this study have also a certain guiding significance for the development of biochip based on glass substrate.

Based on the immobilization enzyme technology and the fluorescence capillary analysis method, the authors have developed a highly sensitive fluorescence reaction system and a novel immobilization multienzyme glucose fluorescence capillary biosensor for determining glucose contents. Reaction principle of the system is that under the catalysis of glucose oxidase (GOD) and horseradish peroxidase (HRP) immobilized on inner surface of a medical capillary, beta-D-glucose reacts with dissolved oxygen to form gluconic acid-delta-lactone and hydrogen peroxide, and then the latter reacts with l-tyrosine to produce a tyrosine dimer, which has maximal excitation and emission wavelengths at 320 nm and 410 nm, respectively. Fluorescence of the dimer is proportional to the concentration of the beta-D-glucose. Optimization conditions suitable for the reaction system and the biosensor were as follows. Concentration of the L-tyrosine used as fluorescence reagent was 0.15 mol L(-1), the active concentrations of the GOD and the HRP for the immobilization were 15 kU L(-1) and 8 kU L(-1), respectively. Consumptions of the sample and reagents in one determination were 5.0 microL and 15 microL, respectively. Quantitative range of the biosensor for the glucose was in the range 1-10 micromol L(-1), its relative standard deviation was less than 4.9%, and its detection limit was 0.62 micromol L(-1). The biosensor's recovery was in the range 96-105%. Results of some serum determined with the biosensor and with a commercial glucose-kit were well coincident to each other. Accordingly, the biosensor can be applied to the determination of serum glucose contents in the diagnosis of diabetes., (Copyright 2009 Elsevier B.V. All rights reserved.)

Goldview marked DNA fluorescence capillary biosensor was studied in the present paper. Based on fluorescence capillary analysis (FCA), the DNA biosensor uses capillary as immobilization carrier and detection carrier of DNA probe. Probes (20-mer-ssDNA) were immobilized on the inner wall of capillary by poly-l-lysine, and DNA fluorescence capillary biosensor (DNA-FCB) was made. After being hybridized with complementary target DNA and dyed by Goldview, the target DNA was qualified or quantified by detecting the fluorescence density of the Goldview using F-4500 spectrofluorometer. The sample volume was 12 microL. The concentration of the target DNA showed good linearity with the fluorescence intensity in the range of 0. 4-4 micromol x L(-1) (2.4-24 mg x L(-1)) (y = 65.911x + 3.9944, r = 0.9989). The RSD was lower than 3.5%. The concentration detection limit of the target DNA was 0.39 micromol x L(-1) (2.2 mg x L(-1)). The DNA-FCB can be used to qualify or quantify the target DNA. It's advantages are simplicity of manipulation, thimbleful of sample and reagent volumes, repeated use of capillary, and the lowest test cost. By using DNA-FCB to qualify the target DNA, we can consumedly decrease the pollution of the environment.

The authors have proposed an immobilized enzymatic fluorescence capillary biosensor (SBAs-IE-FCBS) for the determination of sulfated bile acids (SBAs). The reaction principle of the biosensor is that under the catalysis of the bile acid sulfate sulfatase (BSS) and beta-hydroxysteroid dehydrogenase (beta-HSD) immobilized on inner surface of a medical capillary, SBAs desulfates to 3beta-hydroxyl bile acids, then the latter reacts with nicotinamide adenine dinucleotide (NAD(+)), and is converted into 3-ketosteroid; meanwhile, NAD(+) is converted to reduced nicotinamide adenine dinucleotide (NADH). NADH continuously reacts with 1-methoxy-5-methylphenazinium methyl sulfate (1-MPMS) and is converted into NAD(+) circularly and 1-MPMSH(2). Finally resazurin is reduced into resorufin by 1-MPMSH(2), the formed resorufin (lambda(ex)/lambda(em): 540 nm/580 nm) is used for quantifying the concentration of SBAs. Optimized conditions being suitable with the biosensor are as follows: the concentrations of BSS and beta-HSD used for the immobilization all are 5 kUL(-1); the concentrations of 1-MPMS and resazurin all are 25 micromolL(-1); the concentrations of Tris-HCl buffer and NAD(+) are 100 and 400 micromolL(-1), respectively; total volume of the enzyme, reagent and sample is only 18 microL per time for determining; the reaction temperature is 37 degrees C; the reaction time is 15min. The concentration of SBAs is directly proportional to the fluorescence intensity of the biosensor measured from 0.5 to 5.0 micromolL(-1). The relative standard deviation is less than 3.4%, and the detection limit was 0.16 micromolL(-1). The recoveries are in the range 95.5-106%. This SBA-IE-FCBS can be used for quantifying SBAs in urine to diagnose and judge hepatobiliary diseases, etc.