Liqing Lin

Simultaneous determination of dopamine, ascorbic acid and uric acid at poly (Evans Blue) modified glassy carbon electrode

A sensitive and selective electrochemical method for the determination of dopamine using an Evans Blue polymer film modified on glassy carbon electrode was developed. The Evans blue polymer film modified electrode shows excellent electrocatalytic activity toward the oxidation of dopamine in phosphate buffer solution (pH 4.5). The linear range of 1.0 x 10(-6)-3.0 x 10(-5) M and detection limit of 2.5 x 10(-7) M were observed in pH 4.5 phosphate buffer solutions. The interference studies showed that the modified electrode exhibits excellent selectivity in the presence of large excess of ascorbic acid and uric acid. The separation of the oxidation peak potentials for dopamine-ascorbic acid and dopamine-uric acid were about 182 mV and 180 mV, respectively. The differences are large enough to determine AA, DA and UA individually and simultaneously. This work provides a simple and easy approach to selectively detect dopamine in the presence of ascorbic acid and uric acid in physiological samples.

Electrochemical biosensor based on nanogold-modified poly-eriochrome black T film for BCR/ABL fusion gene assay by using hairpin LNA probe

A novel electrochemical biosensor is described for detection of breakpoint cluster region gene and a cellular abl (BCR/ABL) fusion gene in chronic myelogenous leukemia (CML) by using thiolated-hairpin locked nucleic acids (LNA) as the capture probe. The hairpin LNA probe was immobilized on the nanogold (NG)/poly-eriochrome black T (EBT) film-modified glassy carbon electrode (GCE). The immobilized LNA probe could selectively hybridize with its target DNA on LNA/NG/EBT/GCE surface. The immobilization and hybridization of the LNA probe were characterized with cyclic voltammetry and electrochemical impedance spectroscopy. The hybridization of the immobilized LNA probe with the target DNA was detected by differential pulse voltammetry with the electroactive methylene blue as an indicator. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization, and a high sensitivity. This novel electrochemical biosensor has been used for assay of PCR real sample with satisfactory result.

Ratiometric electrochemical immunoassay based on internal reference value for reproducible and sensitive detection of tumor marker

A ratiometric assay in an electrochemical immunosensor for tumor marker, herein carcinoembryonic antigen (CEA) was chosen as a model analyte, was developed to improve simplicity and accuracy. The immunosensor was fabricated via the simple expedient way of using Polythionine-gold (PTh-Au) as electrode modified material to be an internal reference signal and K3[Fe(CN)6] in electrolyte as an indicator signal. When the CEA was fixed on the modified electrode via immunoreaction, only the indicator signal sensitively altered; by contrast, the internal reference signal of PTh-Au remained constant at a suitable pH of the electrolyte. The ratio between the alterations of the indicator signal of K3[Fe(CN)6] and the constant internal reference signal can be used to monitor the concentration of CEA reliably, reproducibly, and sensitively. The prepared ratiometric electrochemical immunosensor could detect CEA with good specificity within a wide linear range from 0.005ng/ml to 40ng/ml with a detection limit of 2.2pg/ml. Additionally, experimental results confirm that our proposed method is practical. Thus, this method can expand to recognize and test other protein markers.

PRSS1_p.Leu81Met mutation results in autoimmune pancreatitis

AIM To describe protease serine 1 (PRSS1) gene mutations in patients with autoimmune pancreatitis (AIP) and the clinical features of AIP. METHODS Fourteen patients with AIP, 56 with other chronic pancreatitis, 254 with pancreatic cancer and 120 normal controls were studied. The mutations and polymorphisms of four genes involved with pancreatitis or pancreatic cancer, PRSS1, SPINK1, CFTR and MEN1, were sequenced. The pathogenic mechanism of AIP was investigated by comparing the wild-type expression system with the p.81Leu→Met mutant expression system. RESULTS Two novel mutations (p.81Leu→Met and p.91Ala→Ala) were found in PRSS1 gene from four patients with AIP. PRSS1_p.81Leu→Met mutation led to a trypsin display reduction (76.2%) combined with phenyl agarose (Ca(2+) induced failure). Moreover, the ratio of trypsin/amylase in patients with AIP was higher than in the patients with pancreatic cancer and other pancreatitis. A large number of lymphocytes and plasma cells were found in the bile ducts accompanied by hyperplasia of myofibroblasts. CONCLUSION Autoimmune pancreatitis may be related to PRSS1 gene mutations.

Signal-on fluorescent sensor based on GQDs–MnO2 composite for glutathione

Graphene quantum dots (GQDs) exhibit strong photoluminescence, biocompatibility, and low toxicity and can easily functionalize surfaces to improve the detection of biomolecules. As-prepared MnO2 nanosheets were applied in the fabrication of GQDs–MnO2 composite to develop a signal-on fluorescent platform for sensing glutathione (GSH). The fluorescence of the GQDs was quenched by MnO2 in the GQDs–MnO2 composite as a result of fluorescence resonance energy transfer within the composite. The fluorescence of GQDs was restored when GSH was introduced because GSH reduced the MnO2 nanosheets into Mn2+ and released GQDs. Under the optimized conditions, the concentration of GSH can be accurately detected with high sensitivity and specificity in Tris–HCl buffer and simulated serum. The proposed sensing strategy was successfully used to measure GSH in the range of 1–1000 μM with a detection limit of 0.45 μM. In addition, GSH in reduced glutathione injection and reduced glutathione tablets can be determined accurately using the proposed method. GQDs–MnO2 composite is thus a promising GSH fluorescence sensor for the analysis of complex systems and for quality control in the pharmaceutical industry.

A chronocoulometric LNA sensor for amplified detection of K-ras mutation based on site-specific DNA cleavage of restriction endonuclease

An amplified chronocoulometric Locked nucleic acid (LNA) sensor (CLS) for selective electrochemical detection of K-ras mutation was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. Thiolated-hairpin LNA probe with palindrome structure of stem was immobilized on the gold nanoparticles modified gold electrode (NG/AuE). It can be cleaved by EcoRI in the absence of K-ras mutation-type DNA (complementary with the loop part of hairpin probe), but cannot be cleaved in the presence of mutation-type DNA. The difference before and after enzymatic cleavage was then monitored by chronocoulometric biosensor. Electrochemical signals are generated by chronocoulometric interrogation of Hexaammineruthenium (III) chloride (RuHex) that quantitatively binds to surface-confined hairpin LNA probe via electrostatic interactions. The results suggested this CLS had a good specificity to distinguish the K-ras mutation-type, wild-type and non-complementary sequence. There was a good linear relationship between the charge and the logarithmic function of K-ras mutation-type DNA concentration. The detection limit had been estimated as 0.5 fM. It is possible to qualitatively and quantitatively detect K-ras point mutation in pancreatic cancer.

A label-free electrochemical immunosensor based on poly(thionine)–SDS nanocomposites for CA19-9 detection

In this paper, poly(thionine) doped sodium dodecyl sulphate nanocomposites (PThi–SDS NCs) were synthesised for the first time. The morphology and characteristics of the PThi–SDS NCs were investigated by using atomic force microscopy (AFM), UV-vis spectroscopy and electrochemical methods. Owing to the unique structure and properties of the PThi–SDS NC, it was used as a redox electrochemical mediator to construct immunoassay combining gold nanoparticles (AuNPs) as the immobilization matrix for antibody. A novel biomolecular immobilization strategy based on PThi–SDS/AuNPs was used to develop a highly sensitive label-free electrochemical immunosensor for the CA19-9 detection. Under optimal conditions, the present immunosensor exhibited a wide linear range of 5 to 400 U mL−1 with a detection limit of 0.45 U mL−1. Meanwhile, the novel immunosensor exhibited high selectivity, good reproducibility and stability.

Electrocatalytic oxidation and determination of norepinephrine in the presence of ascorbic and uric acids at a poly (Evans Blue)—modified glassy carbon electrode

A sensitive and selective electrochemical method for the determination of norepinephrine using a poly (Evans Blue) film-modified glassy carbon electrode was developed. The polymer film-modified electrode shows excellent electrocatalytic activity toward the oxidation of norepinephrine (NE) in phosphate buffer solution (pH 5.0). The linear range of 5.0 × 10−7–1.8 × 10−5 M and detection limit of 3.5 × 10−8 M were observed for the determination of NE in pH 5.0 phosphate buffer solutions. The interference studies showed that the modified electrode had excellent selectivity for the determination of NE in the presence of large excess of ascorbic acid (AA) and uric acid (UA). The differences of the oxidation peak potentials for NE-AA and NE-UA were about 175 and 172 mV, respectively. The resolution is large enough to determine AA, NE and UA individually. This work provides a simple and easy approach to selective detection of NE in the presence of AA and UA in physiological samples.

Simple and effective label-free electrochemical immunoassay for carbohydrate antigen 19-9 based on polythionine-Au composites as enhanced sensing signals for detecting different clinical samples

Carbohydrate antigen 19-9 (CA19-9) is an important biomarker for the early diagnosis and clinical monitoring of pancreatic cancer. Reliable, simple, and accurate methods for the detection of CA19-9 are still urgently needed. In this study, polythionine-Au composites (AuNPs@ PThi) were designed and prepared through one-pot reaction using HAuCl4 as the co-oxidant and raw material in thionine solution containing FeCl3 as the oxidant. AuNPs@PThi-immobilized glassy carbon electrode was used as a sensitive redox probe for electrochemical interface. AuNPs@PThi not only favored the amplification of electrochemical signals but also facilitated excellent environmental friendliness for bioassay. Maximizing the electrochemical properties of AuNPs@PThi, an effective label-free electrochemical immunoassay for the ultrasensitive and reliable detection of CA19-9 was developed. Under optimal conditions, the linear range of the proposed immunosensor was estimated to range from 6.5 to 520 U/mL, with a detection limit of 0.26 U/mL at a signal-to-noise ratio of 3. The prepared immunosensor for CA19-9 detection showed high sensitivity, stability, and reproducibility. Furthermore, the fabricated immunosensor based on AuNPs@PThi can effectively detect and distinguish clinical serum samples of pancreatic cancer and normal control with accuracy and convenience.

An electrochemical sensor based on DNA polymerase and HRP-SiO2 nanoparticles for the ultrasensitive detection of K-ras gene point mutation

We successfully developed an electrochemical DNA sensor for the precise detection of K-ras gene point mutation. The proposed sensor was based on DNA replication, which fully employed the 5′ → 3′ polymerization activity of DNA polymerase I and the principle of base pairing. The biotin-dUTP only linked with the base A, instead of base G. And then, the streptavidin-HRP-SiO2 nanoparticles combining with the biotin-dUTP on the modified electrode produced the current signal in the TMB solution. The streptavidin-HRP-SiO2 nanoparticles had the effect of amplified signal. Hence, the sensitivity and selectivity of the proposed sensor were observably enhanced, comparison with the previously reported DNA sensors. The response current of the amperometric i–t curve of the prepared sensor showed a good linear correlation with the logarithm of target DNA concentration in the range from 0.001 pM to 1000 pM with the limit of detection of 0.42 fM. And the current value was also proportional to the logarithm of target DNA concentration from 0.01 pM to 100 pM when the hybridization solution contained other mutant K-ras genes and wild K-ras gene. Thus, this proposed approach provided the good basis and a promising platform that the mutant K-ras gene in the clinical genomic sample can be directly detected.