Label-free detection of DNA hybridization using gold-coated tapered fiber optic biosensors (TFOBS) in a flow cell at 1310 nm and 1550 nm

We had previously reported the detection of a model protein bovine serum albumin (BSA) using antibody-immobilized tapered fiber optic biosensors (TFOBS) at 1310 nm and 1550 nm under stagnant and flow conditions. Because of recent interest in pathogen detection based on DNA, in this work we explore the application of these sensors for the detection of single stranded DNA (ssDNA). We show that it is feasible to directly detect the hybridization of a 10-mer ssDNA to its complementary strand immobilized on the sensor surface. Detection was performed under flow conditions because flow reduces non-specific binding to sensor surface, eliminates optical transmission changes due to mechanical movements, and allows for instantaneous switching of samples when needed.

TFOBS were fabricated with waist diameters of 5–10 μm and total lengths of 1000–1200 μm. The taper regions were coated with 50 nm of gold and housed in a specially constructed holder which served as a flow cell. The TFOBS was immobilized with 15-mer ssDNA with a C₆ extension and a thiol group, which attaches to Au1 1 1 sites. Then, the complementary 10-mer ssDNA samples were allowed to flow in from low to high concentration (750 fM to 7.5 nM) and the resulting transmission changes were recorded. It is shown that 750 fM of complementary DNA can be detected. This sensor was able to distinguish between complementary DNA from DNA with a single nucleotide mismatch in the middle position.     

Direct electrochemistry and electrocatalysis with horseradish peroxidase immobilized in polyquaternium-manganese oxide nanosheet nanocomposite films

A novel biocompatible polyquaternium (QY)-manganese oxide nanosheet (MNS) nanocomposite has been prepared and shown to be a promising matrix for horseradish peroxidase (HRP) immobilization. The resulting HRP-QY-MNS film was characterized by Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy, which indicated that HRP retained its native structure in the nanocomposite film. An HRP-QY-MNS film-modified glassy carbon electrode exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks centered at −0.272 V (vs. Ag/AgCl) in pH 7.0 phosphate buffer solution. The direct electrochemical behavior of HRP was greatly enhanced in the QY-MNS nanocomposite film compared with that in single-component QY or MNS films. The immobilized HRP showed excellent electrocatalysis in the reduction of hydrogen peroxide (H₂O₂), which was exploited in the construction of an H₂O₂ biosensor. The linear range of the biosensor for H₂O₂ was found to be from 1.0 × 10⁻⁷ to 3.2 × 10⁻⁵ M with a correlation coefficient of 0.998. The detection limit was 7.8 × 10⁻⁸ M at a signal-to-noise ratio of 3. The biosensor exhibited rapid response and good long-term stability.     

电分析化学在生命科学中的应用

电分析化学在生命科学领域得到广泛的应用,电化学生物传感器是其中之一。它主要包括微生物电极传感器、电化学免疫传感器、组织电极与细胞器电极传感器、酶传感器、电化学DNA传感器等。本人就其发展情况作一概述。     

Enzyme-linked immuno-strip biosensor to detect Escherichia coli O157:H7

A strip-based biosensor using the enzyme-linked immunosorbent assay technique was fabricated to detect Escherichia coli O157:H7. Two types of antibody specified to E. coli O157:H7 were used to form sandwich-binding complexes. To fabricate an immuno-strip, capture antibody (monoclonal antibody) was immobilized onto signal generation pad and polyclonal antibody conjugated with horseradish peroxidase (HRP) was utilized as detection antibody. Four different functional membranes have been used to fabricate immuno-chromatographic assay strip. A sample application pad was a glass fiber membrane pre-treated with polyvinyl alcohol. A conjugate release pad was fabricated using a glass membrane. A signal generation pad was made on nitrocellulose membrane. Finally, a cellulose membrane was used as an absorption pad. Under optimal conditions of analysis, a color signal in proportion to the E. coli O157:H7 concentration was measured using a detector. The measurement range was 1.8×10³–1.8×10⁸ CFU/mL.     

Emerging applications of aptamers to micro- and nanoscale biosensing

Micro- and nanofabrication has allowed the production of ultra-sensitive, portable, and inexpensive biosensors. These devices generally rely on chemical or biological receptors which recognize a particular compound of interest and relay this recognition event effectively by transduction. Recent advances in RNA and DNA synthesis have enabled the use of aptamers, in vitro generated oligonucleotides, which offer high affinity biomolecular recognition to a theoretically limitless variety of analytes. DNA and RNA aptamers have gained so much attention in the biosensor community, that they have begun competing with more established affinity ligands including enzymes, lectins, and most notably, immunoreceptors such as antibodies. This article reviews the current state-of-the-art of aptasensors, or biosensors that use aptamers as molecular recognition elements, emphasizing the synergy between aptamer-based biosensing and micro- and nanotechnology. Aptasensors developed on micro- and nanoscale platforms based on mass changes, electroanalytical techniques, optical transduction, and purification and separation methods will be covered.     

Temperature-insensitive chemical sensor based on a fiber Bragg grating

Temperature-insensitive fiber Bragg grating sensors have been demonstrated by discriminating the peak wavelength difference caused by etching the cladding of half of the grating region. Thermal characteristics and chemical sensitivity of two fiber Bragg gratings are experimentally investigated. Experimental results indicate that the etched half and the rest of the grating region have almost the same thermal response, and concentration changes of the surrounding chemical solutions can be detected by measuring the peak wavelength difference. The two sensors have been used to measure the concentrations of propylene glycol solutions and sugar solutions, and one of them could detect 0.067% and 0.027% concentration change for two kinds of solutions with a low-cost wavelength interrogation module.     

Identification of β-lactam antibiotics using bioluminescent Escherichia coli and a support vector machine classifier algorithm

We propose the use of bioluminescent whole cell biosensor combined with a pattern classification algorithm to automatically detect and identify β-lactam antibiotic substances. Escherichia coli cells with a plasmid harboring luxCDABE genes under the β-lactam sensitive promoter element are used as sensors. We present experimental measurements of light production of bioluminescent bacteria subject to 11 antibiotic substances. The patterns of measured light production are classified using a support vector machine classifier. The accuracy and reliability of the classification suggests that this method can be used in the future to probe for new antibiotic substances.     

A label-free DNA sensor based on impedance spectroscopy

This paper describes a label-free detection system for DNA strands based on gold electrodes and impedance measurements. A single-stranded 18 mer oligonucleotide (ssDNA) was immobilised via a thiol linker on gold film electrodes and served as probe DNA. Residual binding places were filled with mercaptobutanol. The sensor surface clearly distinguished between complementary and non-complementary target ssDNA. Additionally, detection of single base pair mismatches was possible. The electrode was impedimetrically characterised in the presence of the redox system ferri/ferrocyanide before and after DNA hybridisation. Impedance analysis showed that the charge transfer resistance, R_ct, was increasing after DNA duplex formation, whereas the capacitive properties remain rather unaltered. The relative change of R_ct was used as sensor parameter. Concentrations in the nanomolar range have been detected by the system. The sensor was reusable because a denaturation protocol allowed effective double strand dissociation without changing the surface properties of the electrode substantially. The time for DNA detection have been reduced to about 15 min including regeneration. The sensor signal was amplified by about 20% after binding of a negatively charged molecule to the formed DNA duplex. The sensor was also capable of sensing longer target ssDNA strands as shown with 25 mer and 37 mer oligonucleotides.     

重构纳米类脂生物膜制备及敏感机理研究

介绍了重构纳米类脂生物膜(r-NLM)的制备,并采用循环伏安法(CV)研究了温度和外加电场对r-NLM膜的影响,采用“相变温度“及“微桥“模型分析了其原因.从离子载体、离子通道的角度,分析了重构纳米类脂生物膜对不同离子的敏感机理.重构纳米类脂生物膜中离子载体型修饰剂,其不同大小的极性空腔能够络合不同半径的离子,并将离子运输到膜的另一边释放,从而实现对离子的传递;而离子通道型修饰剂则可在膜中形成不同直径的跨膜通道,通道开启时,可进行特定离子的选择性跨膜通道传输.