The enrichment of glycopeptides plays an important role in glycoproteomics. In this paper, a covalent-organic framework called TpPa-1, synthesized by the Schiff base reaction of 1,3,5-triformylphloroglucinol and paraphenylenediamine, was first successfully utilized as a hydrophilic porous material for N-linked glycopeptide enrichment. Using this material, interference from non-glycopeptides could be efficiently eliminated, which facilitated the mass spectrometry detection of glycopeptides. By capturing N-linked glycopeptides from tryptic digests of human IgG, our method was proved to have high sensitivity at the femtomole level. And it showed superior selectivity for glycopeptides even when non-glycopeptides were 1000 times more concentrated. Due to the strong covalent bonds, this material possessed good stability and could be repeatedly used for at least 10 times. The ultra-low mass density and abundant binding sites also provided it with high binding capacity (178 mg g−1, IgG/TpPa-1). Moreover, N-linked glycopeptides were easily enriched by this material from only 10 μL human serum, which demonstrated its potential in pretreatment of complex biological samples.

G-quadruplex sequences exist in eukaryotic organisms and prokaryotes, and the investigation of the interactions between G-quadruplexes and small molecule ligands is important for gene therapy, biosensor fabrication, fluorescence imaging and so on. Here, we investigated the behaviour of methylene blue (MB), an electroactive molecule, in the presence of different intramolecular G-quadruplexes by an electrochemical method using a miniaturized electrochemical device based on its intrinsic electrochemical properties. Although the effects of MB on different intramolecular G-quadruplex structures are not obvious by circular dichroism spectroscopy, distinct differences in the binding affinities of MB with different intramolecular G-quadruplexes were quickly and easily observed by an electrochemical technique. At the same time, for the human telomerase G-rich sequence (HT), the diffusion current of MB changed sensitively under different ionic conditions due to the formation of different conformations of HT, which indicated that our electrochemical method has the potential to study the influence of metal ions on the conformations of the G-quadruplexes with simplicity, rapid response and low cost. From all these, a new stacking mechanism and rule were obtained, which were also validated by docking studies and isothermal titration calorimetry (ITC).

A newly developed sheathless interface for capillary electrophoresis–mass spectrometry, using a porous tip sprayer, was first applied for highly sensitive determination of cytosine modifications. The system performed well in identification and quantification of both 5-methylcytosine and 5-hydroxymethylcytosine using only 125 pg (∼20 cells) genomic DNA samples.

The coupling between open channel-based microchip electrophoresis and mass spectrometry via electrostatic spray ionization is proposed for in situ detection of fractionated analytes. Electrophoretic separation is performed in an open channel fabricated in a plastic substrate. The solvent of background electrolyte is evaporated from the open channel because of Joule heating during electrophoresis, leaving the dried electrophoretic bands to be directly analyzed by mass spectrometry via scanning electrostatic spray ionization. Proof-of-concept results are obtained with fluorescent dyes and antibiotics as the test samples, demonstrating an efficient on-chip detection platform based on the electrophoresis and electrostatic spray ionization mass spectrometry.A novel strategy for microchip electrophoresis and mass spectrometry hyphenation is proposed by in situ electrostatic spray ionization of the electrophoretic bands formed in an open channel.

•The paper focuses on immobilization-free DNA-based homogeneous electrochemical biosensors, aiming at compensating for the deficiency of heterogeneous modes.•The main signal readout modes and signal enhancement strategies will be discussed systematically and the applications combined with miniaturization of electrochemical devices will be specifically introduced.•Immobilization-free DNA-based homogeneous electrochemical biosensors may offer helpful guidance for developing point-of-care assays with potential of commercial applications in many fields such as molecular diagnostics, pharmaceutical screening, drug discovery and so on.DNA is an excellent biomaterial for building biosensors utilizing diverse DNA interactions with a broad range of biomolecules and chemical compounds. DNA-based electrochemical biosensors have been successfully used in numerous applications with advantages of rapid detection, high sensitivity and low cost. Typical DNA-based electrochemical biosensors require an immobilization step of DNA probe as the biological recognition element, which always suffers from time-consuming and labor-intensive electrode pretreatment and immobilization process, expensive chemical modification, lower reactivity, and complicated operation. Immobilization-free DNA-based electrochemical biosensors can provide powerful complementary tools and novel design strategies for traditional electrochemical DNA biosensors. They would be advantageous in some ways, such as low cost, easy operation, and time saving. More importantly, the homogeneous reaction could increase the reliability and reproducibility of electrochemical sensor without the immobilization step. This review introduces multiple immobilization-free strategies for developing DNA-based homogeneous electrochemical biosensors, aiming at compensating for the deficiencies of heterogeneous modes. The main signal readout modes and signal enhancement strategies will be discussed separately. Additionally, the burgeoning applications combined with miniaturization of electrochemical devices will be highlighted.

The construction of compact and robust artificial biochemical circuits based on nucleic acids can help researchers to understand the essential mechanisms of complex biological systems, and design sophisticated strategies for various requirements. In this study, a novel DNA cross-triggered cascading self-amplification artificial biochemical circuit was developed. Once triggered by trace amounts (as low as 2 amol) of either of two fully independent oligonucleotide factors under homogeneous isothermal conditions, the circuit simultaneously amplified both factors by 105–107 fold, which was proved using mass spectrometry. The compact and robust circuit was successfully used to construct a multi-input Boolean logic operation and a sensitive DNA biosensor based on the dual-amplification of both the target and reporter. The circuit showed great potential for signal gain in complicated molecular programming, and flexible control of nucleic acid nanomachines in biochemical network systems and nanotechnology.

Herein, we proposed a portable, easy-to-operate, and antifouling microcapsule array chip for target detection. This prepackaged chip was fabricated by innovative and cost-effective 3D ice printing integrating with photopolymerization sealing which could eliminate complicated preparation of wet chemistry and effectively resist outside contaminants. Only a small volume of sample (2 μL for each microcapsule) was consumed to fulfill the assay. All the reagents required for the analysis were stored in ice form within the microcapsule before use, which guaranteed the long-term stability of microcapsule array chips. Nitrite and glucose were chosen as models for proof of concept to achieve an instant quantitative detection by naked eyes without the need of external sophisticated instruments. The simplicity, low cost, and small sample consumption endowed ice-printing microcapsule array chips with potential commercial value in the fields of on-site environmental monitoring, medical diagnostics, and rapid high-throughput point-of-care quantitative assay.

Taking advantage of the intrinsic characteristics of G-triplet-containing sequences, a pioneering tailor-made clip-like reporter containing three-fourths of a G-quadruplex is established. The reporter can clip the G triplet in the target sequence through a recognition process to form a complete G-quadruplex structure.

Herein, an innovative stable-isotope relative quantification strategy for N-glycans was achieved using a self-designed non-reductive hydrazino-s-triazine deuterated derivative as a labelling reagent combined with mass spectrometry. As much as a 20 Da mass shift could effectively distinguish different forms of N-glycans labelled with normal and heavy hydrazino-s-triazine at the reducing end. Especially for glycans with high molecular weight, qualitative identification could be significantly simplified on account of avoiding isotopic distributions overlapping in the mass spectra. Meanwhile, a deuterium derivative with high purity guaranteed the accuracy of the relative quantification. The obviously undifferentiated response toward normal and heavy hydrazino-s-triazine labelled glycans in electrospray ionization mass spectrometry confirmed the feasibility and reliability of our proposed strategy, which was further demonstrated by relative quantification of the mixtures of labelled N-glycans cleaved from ovalbumin as a model sample. Finally, we adopted human serum as a complex sample and successfully achieved highly accurate detection of 48 N-glycans. The deuterated hydrazino-s-triazine labelling reagent exhibited a great potential to promote the development of highly-efficient, accurate and reliable N-glycan relative quantification in pharmacy and diagnosis research.

A facilely synthesized amino-functionalized metal–organic framework (MOF) MIL-101(Cr)-NH2 was first applied for highly specific glycopeptide enrichment based on the hydrophilic interactions. With the special characteristics of the MOF, the material performed well in selectivity and sensitivity for both standard glycoprotein samples and complex biological samples.

A simple and novel reporter-triggered isothermal exponential amplification reaction (R-EXPAR) integrated with a miniaturized electrochemical device was developed, which achieved excellent improvement (five orders of magnitude) of sensitivity toward reporter, G-quadruplex. This R-EXPAR strategy has been successfully implemented to construct a homogeneous label-free electrochemical sensor for ultrasensitive DNA detection.

Herein, G-quadruplex sequence was found to significantly decrease the diffusion current of methylene blue (MB) in homogeneous solution for the first time. Electrochemical methods combined with circular dichroism spectroscopy and UV–vis spectroscopy were utilized to systematically explore the interaction between MB and an artificial G-quadruplex sequence, EAD2. The interaction of MB and EAD2 (the binding constant, K ≈ 1.3 × 106 M–1) was stronger than that of MB and double-stranded DNA (dsDNA) (K ≈ 2.2 × 105 M–1), and the binding stoichiometry (n) of EAD2/MB complex was calculated to be 1.0 according to the electrochemical titration curve combined with Scatchard analysis. MB was proved to stabilize the G-quadruplex structure of EAD2 and showed a competitive binding to G-quadruplex in the presence of hemin. EAD2 might mainly interact with MB, a positive ligand of G-quadruplex, through the end-stacking with π-system of the guanine quartet, which was quite different from the binding mechanism of dsDNA with MB by intercalation. A novel signal read-out mode based on the strong affinity between G-quadruplex and MB coupling with aptamer/G-quadruplex hairpin structure was successfully implemented in cocaine detection with high specificity. G-quadruplex/MB complex will function as a promising electrochemical indicator for constructing homogeneous label-free electrochemical biosensors, especially in the field of simple, rapid, and noninvasive biochemical assays.

Research on the kinetic characteristics and mechanisms of DNA reactions is crucial for bioengineering and biosensing. A G-quadruplex, which can form a peroxidase-mimicking DNAzyme with hemin, was for the first time used to establish a versatile platform for kinetic investigations on DNA reactions. G-quadruplex sequence EAD2 was incorporated into the corresponding nucleic acid reaction as product. The kinetic curves can be obtained rapidly and simply via the quantification of created DNAzyme. In this paper, the kinetics of isothermal linear strand displacement amplification reactions with different DNA lengths and isothermal exponential amplification reactions were successfully elucidated via the G-quadruplex based monitoring platform. As a safe and accessible alternative to the traditional methods, this robust, label-free, time-saving and high-throughput platform shows great potential for the exploration of more novel DNA reactions or circuits in an ingenious manner.

A novel format of fluorescence immunosorbent assay based on the hybridization chain reaction (HCR) using a DNA intercalating dye for signal readout was constructed for the sensitive detection of targets, both in competitive and sandwich modes. In this platform, the capture and recognition processes are based on immunoreactions and the signal amplification depends on the enzyme-free, isothermal HCR-induced labelling event. After a competitive or a sandwich immunoreaction, a biotinylated capture DNA was bound to a biotinylated signal antibody through avidin, and triggered the HCR by two specific hairpins into a nicked double helix. Gene Finder (GF), a fluorescent probe for double-strand DNA, was intercalated in situ into the amplified chain to produce the fluorescence signal. The limit of detection (LOD) for rabbit IgG in competitive mode by HCR/GF immunoassay was improved at least 100-fold compared with the traditional fluorescence immunoassay using the fluorescein isothiocyanate-labelled–streptavidin or fluorescein isothiocyanate-labelled second antibody as the signal readout. The proposed fluorescence immunoassay was also demonstrated by using α-fetoprotein as the model target in sandwich mode, and showed a wide linear range from 28 ng mL−1 to 20 μg mL−1 with a LOD of 6.0 ng mL−1. This method also showed satisfactory analysis in spiked human serum, which suggested that it might have great potential for versatile applications in life science and point-of-care diagnostics.

•We report a novel G-quadruplex based two-stage isothermal amplification reaction.•The assay achieves dual amplification of recognition event and signal readout.•The strategy realizes simple, rapid, sensitive and label-free DNA colorimetry.•The assay demonstrates good discrimination of mismatched sequences.A novel G-quadruplex based two-stage isothermal exponential amplification reaction (GQ-EXPAR) was developed for label-free DNA colorimetric detection in this work. The exponential amplified trigger DNA in the first stage can convert into G-quadruplex sequence EAD2 by a linear amplification circuit in the second stage. Created EAD2 can form G-quadruplex/hemin DNAzyme to act as a direct signal readout element. The GQ-EXPAR combines the exponential amplification of DNA sequence and the peroxidase-mimicking DNAzyme induced signal amplification, which achieves tandem dual-amplification. Taking advantages of isothermal incubation, this label-free homogeneous assay obviates the need of thermal cycling . As no complex synthesis or extra downstream operation is needed, the whole easy handling procedure can be finished in no more than 1 h. This assay allows the sensing of the model DNA with the limit of detection to be 2.5 pM. Moreover, it demonstrates good discrimination of mismatched sequences. The strategy has also been successfully implemented to sensitively detect Tay–Sachs genetic disorder mutant.

In this study, a simple miniaturized microliter electrochemical device was constructed using a disposable micropipet tip and a reproducible carbon fiber ultramicroelectrode. The novel electrochemical device set the electrochemical reaction in a micropipet tip containing an ultramicroelectrode. We investigated the feasibility of the designed electrochemical device by cyclic voltammetric measurements of redox probe. Its application in an immobilization-free enzyme electrochemical biosensor was also evaluated. Horseradish peroxidase and glucose oxidase were selected to test sensor feasibility. Our results showed that the micropipet tip-based electrochemical device could detect low substrate or enzyme concentration or enzymatic reaction rate. The electrochemical device was applied to analyze the glucose content in human blood samples. With the advantages of low cost, easy operation, rapid detection and high reproducibility, this design provides a new approach in immobilization-free enzyme biosensor construction. Integrated with an ultramicroelectrode, our micropipet tip-based electrochemical device could replace most normal electrodes and electrochemical cells in common laboratories for electroanalysis.

We first developed a label-free and immobilization-free homogeneous electrochemical aptasensor, which combined a smart functional DNA hairpin and a designed miniaturized electrochemical device. Cocaine was chosen as a model target. The anticocaine aptamer and peroxidase-mimicking DNAzyme were integrated into one single-stranded DNA hairpin. Both aptamer and G-quadruplex were elaborately blocked by the stem region. The conformation switching induced by the affinity interaction between aptamer and cocaine released G-quadruplex part and turned on DNAzyme activity. The designed electrochemical device, constructed by a disposable micropipet tip and a reproducible carbon fiber ultramicroelectrode, was applied to the detection of homogeneous DNAzyme catalytic activity at the microliter level. The aptasensor realized the quantification of cocaine ranging from 1 to 500 μM with high specificity. The clever combination of the functional DNA hairpin and the novel device achieved an absolutely label-free electrochemical aptasensor, which showed excellent performance like low cost, easy operation, rapid detection, and high repeatability.

A compatible buffer system for coupling of capillary electrophoresis (CE) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was developed. The employed interface consists of a robot to drive a silver-covered separation capillary and an AnchorChip MALDI-MS target. The outlet of the capillary is grounded and connected to the pre-deposited buffer droplet on the MALDI target to make the electric connection and allow sample crystallization for MALDI-MS. The possibility of using only one buffer already containing the matrix for MALDI-MS for separation and ionization was investigated and tested on protein and peptide samples. The results show that the proposed buffer system is suitable for CE-MALDI-MS coupling, simplifies the traditional buffer mixing steps in off-line CE-MALDI-MS protocols, and is therefore highly promising for on-line analysis.

Tetrahydrogestrinone and related anabolic androgenic steroids (AASs) have been used extensively for performance enhancement (doping) in sports due to their ability to accelerate protein synthesis and improve physical performance (F. C. W. Wu, Clin. Chem., 1997, 43, 1289–1292). The separation of these AASs was performed by micellar electrokinetic chromatography (MEKC) using borate/borax buffer (200/50 mM, pH 8.6), with sodium cholate (40 mM) as a chiral additive. The analytes included two endogenous steroid hormones (testosterone and epitestosterone) and five synthetic anabolic steroids (methyltestosterone, nandrolone, gestrinone, dihydrogestrinone and tetrahydrogestrinone). Capillary electrophoresis parameters such as the concentration of additives, injection time, temperature, and applied voltage were investigated to improve the separation efficiency. A complete separation was achieved in less than 16 min under the optimized conditions. The RSDs of peak area and migration time were below 3.9% and 0.34%, respectively. The limit of detection (LOD) between 240 and 570 ng mL−1 was obtained for each of the pure standards with a photodiode array (PDA) detector. Lower LODs could be reached when combining with preconcentration. After liquid–liquid extraction, the recoveries of spiked urine samples were in the range from 88% to 99%.

Enzyme linked aptamer assay (ELAA) uses an aptamer as recognition element and enzyme as signal readout element for establishing different kinds of aptasensors. We reported herein a high-throughput colorimetric aptasensor based on ELAA only requiring a single aptamer sequence for cocaine detection. An anti-cocaine aptamer was cleaved into two fragments, one of which was immobilized on a DNA-BIND 96-well plate via 5′-labeled primary amine and the other one was biotin labeled. The presence of two aptamer fragments and the target molecule led to the formation of aptamer fragments/target complexes. Streptavidin–horseradish peroxidase (SA–HRP) was used to react with biotin in order to obtain quantitative signals. A linear response towards cocaine concentration in the range of 5–200 μM and a detection limit down to 2.8 μM (S/N=3) were achieved. The specificity and application in real sample were validated. Furthermore, a verification test of thrombin detection in the same strategy illustrated its feasibility for not only small molecule but also biomacromolecule. With the advantage of high-throughput, easy operation, high specificity, the colorimetric assay based on ELAA requiring a single aptamer sequence opens up a new approach for detecting different kinds of targets via specific affinity recognition among target and suitably cleaved aptamer fragments.Highlights► High-throughput colorimetric aptasensor based on ELAA is established. ► Novel ELAA type using only a single aptamer was introduced into microplate. ► Detection is based on self-assembling aptamer fragment/target complex. ► The model is successfully used to detect cocaine and thrombin. ► It is a powerful platform to detect different kinds of targets.

•We report a functional DNA hairpin with aptamer and peroxidase-mimicking DNAzyme.•A fill-in-the-blank process is developed for sequence design.•The biosensor can realize rapid and simple “mix-and-measure” detection.•It is the first label-free hairpin for homogeneous colorimetric cocaine detection.The combination of aptamer and peroxidase-mimicking DNAzyme within a hairpin structure can form a functional DNA probe. The activities of both aptamer (as biorecognition element) and DNAzyme (as signal amplification element) are blocked via base pairing in the hairpin structure. The presence of target triggers the opening of the hairpin to form target/aptamer complex and releases G-quadruplex sequence which can generate amplified colorimetric signals. In this work, we elaborated a universal and simple procedure to design an efficient and sensitive hairpin probe with suitable functional DNA partners. A fill-in-the-blank process was developed for sequence design, and two key points including the pretreatment of the hairpin probe and the selection of suitable signal transducer sequence were proved to enhance the detection sensitivity. Cocaine was chosen as a model target for a proof of concept. A series of hairpins with different numbers of base pairs in the stem region were prepared. Hairpin-C10 with ten base pairs was screened out and a lowest detectable cocaine concentration of 5 μM by colorimetry was obtained. The proposed functional DNA hairpin showed good selectivity and satisfactory analysis in spiked biologic fluid. The whole “mix-and-measure” detection based on DNA hairpin without the need of immobilization and labeling was indicated to be time and labor saving. The strategy has potential to be transplanted into more smart hairpins toward other targets for general application in bioanalytical chemistry.

In this work, a novel labelling reagent with multi-charges was designed and developed for glycans capillary electrophoresis-mass spectrometry (CE-MS) analysis. Glycans labelled with this reagent have multiple charges. The results showed that the separation efficiencies of glycans were improved and the sensitivities of large glycans were enhanced more than 10 fold.

An aptamer is an artificial functional oligonucleic acid, which can interact with its target molecule with high affinity and specificity. Enzyme linked aptamer assay (ELAA) is developed to detect cocaine using aptamer fragment/cocaine configuration based on the affinity interaction between aptamer fragments with cocaine. The aptasensor was constructed by cleaving anticocaine aptamer into two fragments: one was assembled on a gold electrode surface, while the other was modified with biotin at 3′-end, which could be further labelled with streptavidin-horseradish peroxidase (SA-HRP). Upon binding with cocaine, the HRP-labelled aptamer fragment/cocaine complex formed on the electrode would increase the reduction current of hydroquinone (HQ) in the presence of H2O2. The sensitivity and the specificity of the proposed electrochemical aptasensor were investigated by differential pulse voltammetry (DPV). The results indicated that the DPV signal change could be used to sensitively detect cocaine with the dynamic range from 0.1 μM to 50 μM and the detection limit down to 20 nM (S/N = 3). The proposed aptasensor has the advantages of high sensitivity and low background current. Furthermore, a new configuration for ELAA requiring only a single aptamer sequence is constructed, which can be generalized for detecting different kinds of targets by cleaving the aptamers into two suitable segments.Highlights► A simple and sensitive aptasensor for cocaine detection based on enzyme linked aptamer assay (ELAA) is developed. ► The detection limit down to 20 nM for cocaine has been achieved by thereaction of H2O2 and HQ catalyzed by HRP. ► The aptasensor shows the potential application in biological fluid. ► A new configuration for ELAA requiring only a single aptamer sequence can be generalized to detect different kinds of targets.

A simple and label-free aptasensor for sensitive and specific detection of cocaine was developed by measuring the change in electrochemical impedance spectra (EIS), based on the formation of a supramolecular aptamer fragments/substrate complex. An anticocaine aptamer was divided into two fragments, Cx and Cy. Three different sensing interfaces, called Au/Cx5S/MCE, Au/Cy3S/MCE and Au/Cy5S/MCE, were fabricated by immobilizing Cx or Cy on a gold electrode through modifying their 5′ or 3′ end with a thiolated group followed by the treatment with mercaptoethanol (MCE). The formation of the corresponding supramolecular aptamer fragments/cocaine complex was investigated via monitoring electrochemical impedance spectra in the presence of [Fe(CN)6]3−/4−. The interfacial electron transfer resistance (Ret) was found to depend strongly on the cocaine concentration. Since the supramolecular aptamer fragments/cocaine complex was formed on the electrode surface, the sensing interface strongly affected the sensitivity of the aptasensor. Au/Cx5S/MCE was shown to have good sensitivity within a cocaine detection range of 0.1–20 μM. Moreover, MCE was shown to improve the sensitivity of the aptasensor greatly. Even without the help of amplification or labeling, cocaine concentrations as low as 100 nM could be easily detected by the impedimetric aptasensor developed. The specificity and regeneration of the cocaine aptasensor were also investigated and satisfactory results were obtained. The developed aptasensor was successfully applied to detect the cocaine in biological fluids.Highlights► A simple label-free aptasensor was developed by electrochemical impedance spectra. ► For the optimization, we designed and compared three different sensing interfaces. ► The sensor could detect as low as 100 nM cocaine without amplification or labeling. ► The aptasensor showed the potential application in biological fluid.

The separation and sequencing of DNA are the main objectives of the Human Genome Project, and this project has also been very useful for gene analysis and disease diagnosis. Capillary electrophoresis (CE) is one of the most common techniques for the separation and analysis of DNA. DNA separations are usually achieved using capillary gel electrophoresis (CGE) mode, in which polymer gel is packed into the capillary. Compared with a traditional CGE matrix, a hydrophilic polymer matrix, which can be adsorb by the capillary wall has numerous advantages, including stability, reproducibility and ease of automation. Various water-soluble additives, such as linear poly(acrylamide) (PAA) and poly(N,N-dimethylacrylamide) (PDMA), have been employed as media. In this study, different star-shaped PDMA polymers were designed and synthesized to achieve lower polymer solution viscosity. DNA separations with these polymers avoid the disadvantages of high viscosity and long separation time while maintaining high resolution (10 bp between 271 bp and 281 bp). The influences of the polymer concentration and structure on DNA separation were also determined in this study; higher polymer concentration yielded better separation performance, and star-like polymers were superior to linear polymers. This work indicates that modification of the polymer structure is a potential strategy for optimizing DNA separation.

A novel matrix, gold nanoparticles–bacterial cellulose nanofibers (Au–BC) nanocomposite was developed for enzyme immobilization and biosensor fabrication due to its unique properties such as satisfying biocompatibility, good conductivity and extensive surface area, which were inherited from both gold nanoparticles (AuNPs) and bacterial cellulose nanofibers (BC). Heme proteins such as horseradish peroxidase (HRP), hemoglobin (Hb) and myoglobin (Mb) were successfully immobilized on the surface of Au–BC nanocomposite modified glassy carbon electrode (GCE). The immobilized heme proteins showed electrocatalytic activities to the reduction of H2O2 in the presence of the mediator hydroquinone (HQ), which might be due to the fact that heme proteins retained the near-native secondary structures in the Au–BC nanocomposite which was proved by UV–vis and IR spectra. The response of the developed biosensor to H2O2 was related to the amount of AuNPs in Au–BC nanocomposite, indicating that the AuNPs in BC network played an important role in the biosensor performance. Under the optimum conditions, the biosensor based on HRP exhibited a fast amperometric response (within 1 s) to H2O2, a good linear response over a wide range of concentration from 0.3 μM to 1.00 mM, and a low detection limit of 0.1 μM based on S/N = 3. The high performance of the biosensor made Au–BC nanocomposite superior to other materials as immobilization matrix.

Orientedly bioconjugated core/shell Fe3O4@Au magnetic nanoparticles were synthesized for cell separation. The Fe3O4@Au magnetic nanoparticles were synthesized by reducing HAuCl4 on the surfaces of Fe3O4 nanoparticles, which were further characterized in detail by TEM, XRD and UV–vis spectra. Anti-CD3 monoclonal antibody was orientedly bioconjugated to the surface of Fe3O4@Au nanoparticles through affinity binding between the Fc portion of the antibody and protein A that covalently immobilized on the nanoparticles. The oriented immobilization method was performed to compare its efficiency for cell separation with the non-oriented one, in which the antibody was directly immobilized onto the carboxylated nanoparticle surface. Results showed that the orientedly bioconjugated Fe3O4@Au MNPs successfully pulled down CD3+ T cells from the whole splenocytes with high efficiency of up to 98.4%, showing a more effective cell-capture nanostructure than that obtained by non-oriented strategy. This developed strategy for the synthesis and oriented bioconjugation of Fe3O4@Au MNPs provides an efficient tool for cell separation, and may be further applied to various fields of bioanalytical chemistry for diagnosis, affinity extraction and biosensor.

Al2O3-modified Li(Ni1/3Co1/3Mn1/3)O2 is synthesized by a modified Al2O3 coating process. The Al2O3 coating is carried out on an intermediate, (Ni1/3Co1/3Mn1/3)(OH)2, rather than on Li(Ni1/3Co1/3Mn1/3)O2. As a comparison, Al2O3-coated Li(Ni1/3Co1/3Mn1/3)O2 also is prepared by traditional Al2O3 coating process. The effects of Al2O3 coating and Al2O3 modification on structure and electrochemical performance are investigated and compared. Electrochemical tests indicate that cycle performance and rate capability of Li(Ni1/3Co1/3Mn1/3)O2 are enhanced by Al2O3 modification without capacity loss. Al2O3 coating can also enhance the cycle performance but cause evident capacity loss and decline of rate capability. The effect of Al2O3 coating and Al2O3 modification on kinetics of lithium-ion transfer reaction at the interface of electrode/electrolyte is investigated via electrochemical impedance spectra (EIS). The result support that the Al2O3 modification increase Li+ diffused coefficient and decrease the activation energy of Li+ transfer reaction but the traditional Al2O3 coating lead to depression of Li+ diffused coefficient and increase of activation energy.

A pseudo-homogeneous immunoextraction method based on gold-coated magnetic nanoparticles (MNPs) for the specific extraction and quantitative analysis of epitestosterone (17α-hydroxy-4-androsten-3-one, abbreviated as “ET”) from human urine samples by high-performance liquid chromatography (HPLC) has been developed. Half-IgG of anti-ET monoclonal antibodies were covalently immobilized onto (Fe3O4)core-Aushell (Fe3O4@Au) MNPs. An external magnetic field was applied to collect the MNPs which were then rinsed with distilled water followed by elution with absolute methanol to obtain ET as the analyte. The obtained extraction solution was analyzed by HPLC with UV detection (244 nm) within 12 min. The standard calibration curve for ET showed good linearity in the range of 20–200 ng mL−1 in phosphate-buffered saline (PBS) solutions with acceptable accuracy and precision. Limit of detection for ET was 0.06 ng mL−1 due to an enrichment factor of 100-fold was achieved. The results obtained by the present method for spiked human urine samples were in agreement with those from indirect competitive enzyme-linked immunoadsorbent assays (ELISAs). The antibody-conjugated Fe3O4@Au MNPs are novel materials for immunoaffinity extraction. Compared with the conventional technique using immunoaffinity column, the method described here for sample pretreatment was fast, highly specific, and easy to operate.

Epitestosterone (ET) has been used as a masking agent and prohibited by the World Anti-Doping Agency (WADA) because its administration will decrease the urinary T/ET ratio, a marker of testosterone (T) administration. In this study, an off-line immunoaffinity extraction coupled with high performance liquid chromatography (HPLC) was developed to quantify the endogenous steroid ET in human urine. The immunoaffinity column (IAC) was prepared by immobilizing the anti-ET monoclonal antibodies on CNBr-activated Sepharose 4B, which can remove the contaminations and non-target compounds from matrix to enrich the target analyte ET. The mobile phase was ammonium acetate (10 mM, pH 4.0)/acetonitrile (45/55, v/v) at an isocratic flow of 1.0 mL/min and the UV absorbance detection wavelength was 244 nm for the detection of ET. The IAC showed good reliability and durability since it had been used for more than 100 runs in a year. The limit of quantification (LOQ) was 1 ng/mL. Satisfied repeatability and precision of the day-to-day and within-day were obtained with the RSD values less than 10%. Results of the recovery of the urine samples were ranged from 98% to 102% with repeatability less than 9%, indicating that the method developed can be used for the real urine sample analysis.

Testosterone is one of the most common doping drugs abused by athletes. Therefore, it is necessary to develop a sensitive and simple method to monitor testosterone and its epimer epitestosterone. An off-line immunoaffinity extraction followed by capillary electrophoresis for simultaneous determination of testosterone and epitestosterone has been described in this paper. Anti-epitestosterone monoclonal antibody which is specific to both testosterone and epitestosterone had been prepared and immobilized on a Sepharose 4B stationary phase. The immunoaffinity column was used for sample cleanup, extraction and preconcentration. After elution and reconstitution, testosterone and epitestosterone in the sample were separated and quantified by micellar electrokinetic chromatography(MEKC) using the borate buffer (200 mM borate, pH 8.7) containing 40 mM sodium cholate as a chiral selector. The immunoaffinity column was evaluated in different parameters such as the retention mechanism, selectivity, binding capacity, elution protocol, and reusability. The separation of these two compounds by MEKC was also optimized. Limit of detection for testosterone and epitestosterone were 5 and 23 ng mL−1, respectively. It was satisfactory to apply this method to analyze testosterone and epitestosterone in spiked urine sample with the recoveries from 78% to 109%.

An immunoaffinity capillary column has been made with poly (2-vinyl-4, 4-dimethylazlactone-co-2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (VDA-co-HEMA-co-EDMA) monolith as a support for the immobilized antibody. The monolith is prepared by UV-initiated in situ polymerization, followed by immobilizing anti-testosterone polyclonal antibody through the rapid reaction with VDA. Fluorescence labeled testosterone at C3 is designed as a tracer to estimate the extraction ability of this immunoaffinity column, and to optimize the immunoextraction process, such as washing, eluting, incubation and injection. The performance in a more realistic application is then demonstrated successfully for the rapid extraction of testosterone (T) by competitive immunoassay and on-line laser-induced fluorescence (LIF) detection. This immunoaffinity monolith is easily prepared, with high mechanical strength and low flow resistance. It is a satisfactory material as an immunoextractor to detect small molecular compounds specifically and rapidly with high sensitivity (sub ng/mL level).

A magnetic beads based immunoaffinity capillary electrophoresis method for total Immunoglobulin E quantification in serum has been developed. The method combines speed, automation ability, and minimal sample consumption. Only 1 μL of serum is required while the whole immunoaffinity capillary electrophoresis method is performed in less than 50 min. The concomitant use of online immunocapture, transient isotachophoresis, and laser-induced fluorescence detection provides a sensitivity in the low picomolar range and a highly linear fluorescence response over 4 orders of magnitude (IgE concentration ranging from 2.4 to 2400 ng/mL). After validation with a reference material, the method has been successfully applied to the quantification of total IgEs in patient sera. The results compared well with classical ImmunoCap data.

The interactions between fluoroquinolones and human serum albumin (HSA) were investigated by affinity capillary electrophoresis (ACE) and fluorescence quenching technique. Based on the efficient separation of several fluoroquinolones using a simple phosphate buffer, the binding constants of fluoroquinolones with HSA were determined simultaneously during one set of electrophoresis by ACE method. The thermodynamic parameters were obtained from data at different temperatures, and the negative ΔH and ΔS values showed that both hydrogen bonds and van der Waals interaction played major roles in the binding of fluoroquinolones to HSA. The interactions were also studied by fluorescence quenching technique. The results of fluorescence titration revealed that fluoroquinolones had the strong ability to quenching the intrinsic fluorescence of HSA through the static quenching procedure. The binding site number n, apparent binding constant Kb and the Stern–Volmer quenching constant Ksv were determined. The thermodynamic parameters were also studied by fluorescence method, and the results were consonant with that of ACE.

Novel fluorescent YVO4:Eu nanocrystals were ∼15 nm in diameter and had been functionalized with phosphorous polyacrylic acids at the surface during the synthesis, which illuminated their potential in bioassays. In this paper, bovine serum albumin (BSA) was covalently coupled to activated nanocrystals from primary amine groups by EDC and sulfo-NHS chemistry successfully. Reaction condition, including concentration, pH value and separation methods, were carefully chosen and well discussed. Binding site number was determined simply by measuring BSA concentration using optimized microplate bicinchonic acid (BCA) assay and nanocrystals concentration according to their fluorescence emission in the monodisperse bioconjugate suspension. Transmission electron microscopy (TEM) imaging showed the monodispersity of the bioconjugates. Fluorescent characteristics were also retained. Immunoblots verified that BSA was covalently coupled to nanocrystals and remained immunoreactive. Finally, the two-step heterogeneous time-resolved fluoroimmunoassay (TR-FIA) of rabbit anti-BSA polyclonal antibody (PcAb) was operated to further validate the bioconjugates.

A novel multi-target antibody to morphine and derivatives was induced by designed morphine-3-site substituted and the polyclonal antibody was prepared with immunizing rabbits. A simple, specific and accurate method for the determination of morphine and related compounds, codeine, acetylcodeine, 6-monoacetylmorphine and morphine-3-glucuronide in urine of heroin abusers, has been developed using the multi-target immunoaffinity column (IAC) prior to capillary electrophoresis separation. The analytes were extracted from the urine of drug addicts with the column, which was made by coupling CNBr-activated Sepharose 4B and multi-target polyclonal antibodies. The analytes of interest were extracted with a methanol/water mixture in one step. Baseline separation of these analogs was achieved by CE using β-cyclodextrin as additive and they were monitored at 214 nm. The assay presented very good reproducibility and precision with the recovery and detection limit between 91–105% and 10–20 ng/mL based on S/N = 2, respectively. The inter-day and intra-day variation, capacity and elution conditions of the immunoaffinity column were also discussed. The metabolites in five heroin addicts’ urine were measured by the present method. The experimental results indicated that the combination of IAC and CE was a useful technique for determination of heroin metabolites from urine samples.

The interaction and binding abilities between the trans-activation response (TAR) element, residues 1–59 of the human immunodeficiency virus type 1 (HIV-1) mRNA, and four computer-simulated designed potentially small-molecule drugs were studied by capillary zone electrophoresis (CZE). Experimental results indicated that these computer-simulated small-molecule drugs could specifically bind to TAR RNA and effectively inhibit the complex formation of HIV-1 trans-activator of transcription (Tat) protein to TAR RNA – an essential component for HIV-1 transcription. The small molecular inhibitors and TAR RNA could be baselinely separated for each individual drug under optimized CE experimental conditions. Their binding constants were quantitatively determined and the data were used for the drug evaluation and screen in lead discovery.

3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been demonstrated to be a key target for drug design against SARS. The interaction between SARS coronavirus 3C-like (3CL) proteinase and an octapeptide interface inhibitor was studied by affinity capillary electrophoresis (ACE). The binding constants were estimated by the change of migration time of the analytes in the buffer solution containing different concentrations of SARS 3CL proteinase. The results showed that SARS 3CL proteinase was able to complex with the octapeptide competitively, with binding constants of 2.44 × 104 M−1 at 20 °C and 2.11 × 104 M−1 at 37 °C. In addition, the thermodynamic parameters deduced reveal that hydrophobic interaction might play major roles, along with electrostatic force, in the binding process. The ACE method used here could be developed to be an effective and simple way of applying large-scale drug screening and evaluation.

The study on the interactions between two anti-human immunodeficiency virus type 1 (anti-HIV-1) active compounds with trans-activation response (TAR) RNA by affinity capillary electrophoresis (ACE) with UV absorbance detection is presented. The results showed that the novel active molecules could interact with TAR RNA and inhibit the reproduce process of HIV-1. The binding constants were estimated by the change of migration time of the analytes through the change of concentrations of TAR RNA in the buffer solution. The yielded binding constants of 8.87 × 103 M−1 for active compound C3 and 8.42 × 103 M−1 for MC3 at 20.0 °C, 0.626 × 103 M−1 and 0.644 × 103 M−1 at 37.0 °C, respectively. The thermodynamic parameters ΔH and ΔS were obtained and shown that both hydrophobic and electrostatic interaction played roles in the binding processes. The results showed that the presented method was an easy and simple method to evaluate the interaction of small molecules with some bioactive materials.

Tetrahydrogestrinone and related anabolic androgenic steroids (AASs) have been used extensively for performance enhancement (doping) in sports due to their ability to accelerate protein synthesis and improve physical performance (F. C. W. Wu, Clin. Chem., 1997, 43, 1289–1292). The separation of these AASs was performed by micellar electrokinetic chromatography (MEKC) using borate/borax buffer (200/50 mM, pH 8.6), with sodium cholate (40 mM) as a chiral additive. The analytes included two endogenous steroid hormones (testosterone and epitestosterone) and five synthetic anabolic steroids (methyltestosterone, nandrolone, gestrinone, dihydrogestrinone and tetrahydrogestrinone). Capillary electrophoresis parameters such as the concentration of additives, injection time, temperature, and applied voltage were investigated to improve the separation efficiency. A complete separation was achieved in less than 16 min under the optimized conditions. The RSDs of peak area and migration time were below 3.9% and 0.34%, respectively. The limit of detection (LOD) between 240 and 570 ng mL−1 was obtained for each of the pure standards with a photodiode array (PDA) detector. Lower LODs could be reached when combining with preconcentration. After liquid–liquid extraction, the recoveries of spiked urine samples were in the range from 88% to 99%.

A compatible buffer system for coupling of capillary electrophoresis (CE) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was developed. The employed interface consists of a robot to drive a silver-covered separation capillary and an AnchorChip MALDI-MS target. The outlet of the capillary is grounded and connected to the pre-deposited buffer droplet on the MALDI target to make the electric connection and allow sample crystallization for MALDI-MS. The possibility of using only one buffer already containing the matrix for MALDI-MS for separation and ionization was investigated and tested on protein and peptide samples. The results show that the proposed buffer system is suitable for CE-MALDI-MS coupling, simplifies the traditional buffer mixing steps in off-line CE-MALDI-MS protocols, and is therefore highly promising for on-line analysis.

A newly developed sheathless interface for capillary electrophoresis–mass spectrometry, using a porous tip sprayer, was first applied for highly sensitive determination of cytosine modifications. The system performed well in identification and quantification of both 5-methylcytosine and 5-hydroxymethylcytosine using only 125 pg (∼20 cells) genomic DNA samples.

In this work, a novel labelling reagent with multi-charges was designed and developed for glycans capillary electrophoresis-mass spectrometry (CE-MS) analysis. Glycans labelled with this reagent have multiple charges. The results showed that the separation efficiencies of glycans were improved and the sensitivities of large glycans were enhanced more than 10 fold.

The construction of compact and robust artificial biochemical circuits based on nucleic acids can help researchers to understand the essential mechanisms of complex biological systems, and design sophisticated strategies for various requirements. In this study, a novel DNA cross-triggered cascading self-amplification artificial biochemical circuit was developed. Once triggered by trace amounts (as low as 2 amol) of either of two fully independent oligonucleotide factors under homogeneous isothermal conditions, the circuit simultaneously amplified both factors by 105–107 fold, which was proved using mass spectrometry. The compact and robust circuit was successfully used to construct a multi-input Boolean logic operation and a sensitive DNA biosensor based on the dual-amplification of both the target and reporter. The circuit showed great potential for signal gain in complicated molecular programming, and flexible control of nucleic acid nanomachines in biochemical network systems and nanotechnology.

A simple and novel reporter-triggered isothermal exponential amplification reaction (R-EXPAR) integrated with a miniaturized electrochemical device was developed, which achieved excellent improvement (five orders of magnitude) of sensitivity toward reporter, G-quadruplex. This R-EXPAR strategy has been successfully implemented to construct a homogeneous label-free electrochemical sensor for ultrasensitive DNA detection.

A facilely synthesized amino-functionalized metal–organic framework (MOF) MIL-101(Cr)-NH2 was first applied for highly specific glycopeptide enrichment based on the hydrophilic interactions. With the special characteristics of the MOF, the material performed well in selectivity and sensitivity for both standard glycoprotein samples and complex biological samples.