Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.

Simple Quantitation of Flucytosine in Low Volume Serum Samples and Evaluation of Common Endogenous Interferences by HPLC-UV

5-Fluorocytosine (5-FC) is used for the treatment of several infections. It is extremely important to monitor blood level concentration for maximum activity to avoid its side effects. A simple, faster, and more accurate analytical method is developed and validated using high-performance liquid chromatography with UV detection in a very low-volume serum sample. Exactly 50 μL of serum was precipitated with 5% trichloroacetic acid. After mixing and centrifugation, 20 μL of supernatant was injected into the HPLC column. Detection was performed at 280 nm. The method is very specific and free from interfering substances due to different drugs and their different circulating metabolites. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.50 μg/L and 1.0 μg/L, respectively. The method was linear in the range of 5 - 150 μg/L in the serum sample. In method comparison, the correlation coefficient r2 was 0.999 and the percentage recovery was 90% - 105% on four levels of the quality control samples. Within run and between run precision was found to be less than 2.2% at four different concentrations (5, 25, 50, and 100 μg/L). A simple, faster, and more accurate HPLC-UV method is developed which is very useful for monitoring 5-FC concentration in low volume serum samples without evaporation step and ion exchange chromatography within minutes.

Molecular recognition triggered aptazyme cascade for ultrasensitive detection of exosomes in clinical serum samples

Exosomes have attracted widespread interest due to their inherent advantages in tumor diagnosis and treatment monitoring.However,it is still a big challenge for highly sensitive and specific detection of exosome in real complexed samples.Herein,a molecular recognition triggered aptazyme cascade strategy was developed for ultrasensitive detection of cancer exosomes in clinical serum samples.In this design,one target exosome could capture a large quantity of aptazymes for the first-step signal amplification.And then the captured aptazyme was activated and recycled to release the fluorophore-labelled substrate strand for a cascaded signal amplification.Notably,the activation of aptazyme only occurs whenithas bound with target exosome,ensuring a low background.The experimental results show that the limit of detection(LOD)and the limit of quantification(LOQ)are 3.5×10^(3) particles/μL and 1.7×10^(4) particles/μL,respectively,which is comparable to the results of most existed fluorescence-based exosome probes.Moreover,this assay possesses high specificity to distinguish exosomes derived from other cell lines.Furthermore,this fluorescence probe was utilized in cancer patient and healthy serum samples successfully,suggesting its great potential for clinical diagnosis and biological studies.

Electrochemical Determination of Alkaline Phosphatase in Human Serum by Differential Pulse Voltammetry

Differential pulse voltammetry(DPV) was applied to the determination of alkaline phosphatase(ALP) activity in human serum with phenyl phosphate as the substrate. Phenyl phosphate can enzymatically be hydrolyzed to produce phenol which is quantified by DPV at +660 mV(vs.Ag/AgCl) in the concentration range of 2.0_100 μmol/L. The standard curve for ALP is linear over the range from 0.06 to 1000 U/L with a relative standard deviation of 3.0%. The conditions for the enzymatic reaction and voltammetric detection were optimized and the kinetic constants were also examined.The human serum samples were tested by this method and the results were in good agreement with those obtained by the routine p-nitrophenyl phosphate spectrophotometric method.

A novel luminol-based chemiluminescence method for the determination of amikacin sulfate in serum by using trivalent copper-periodate complex

A novel chemiluminescence （CL） reaction was based on the oxidizing reaction of luminol by the trivalent copper-periodate complex （Ks[Cu（HIO6）2], DPC） in alkaline medium. The CL intensity could be enhanced in the presence of amikacin sulfate （AKS）. A new CL method was developed for the determination of AKS by coupling with flow injection （FI） technology. Because of the distinctive oxidative effect of DPC, the luminol-based CL reaction could occur at a low concentration of 10-7 M. The relative CL intensity was proportional to the concentration of AKS in the range of 4.0 x 10-9-4.0 x 10-6 g/mL with the detection limit of 1.2 x 10-9 g/mL. The relative standard deviation was 2.1% for 8.0xl0-9g/mL AKS （n=9）. The proposed method was successfully applied to the direct determination of AKS at the level of ng/mL in serum samples. The recovery varied from 97.0% to 106.3%. A possible mechanism of the CL reaction was discussed in detail by relating to the CL kinetic characteristics and electrochemical activities of the oxidant DPC.

Electrochemical Study on the Interaction of Protein with Bromothymol Blue and Its Analytical Application

The interaction of bromothymol blue（BB） with human serum albumin（HSA） was studied by electrochemical techniques and a sensitive method for proteins assay was developed. When BB interacted with HSA, the voltammetric peak current value of BB decreased linearly with the concentration of HSA in a range of 1.0--40.0 mg/L, and the peak potential shifted negatively. Based on the results, a sensitive assay method for proteins, such as HSA, bovine serum albumin（BSA）, and egg albumin etc. was established. This method was further applied to determining the HSA in healthy human blood samples, and the results are not significantly different from those obtained by the classic Coomassie Brilliant Blue G-250 spectrophotometic method. The detecting conditions of this method were optimized and the interaction mechanism was discussed. The results show that the electrochemical parameters（formal potential E^0, standard rate constant of the electrode reaction ks, parameter of kinetic nα） of BB have no obvious changes before and after the interaction, which indicate that BB can interact with HSA, forming an electrochemical non-active complex. The equilibrium constant（βs） and the binding ratio（m） for this complex were calculated. The m is 4 and βs is 1.41 × 10^19. This method is fast, simple, highly sensitive, and has good selectivity, which can be used in clinical measurements.