Expression and purification of soluble recombinant ß-lactamases using Escherichia coli as expression host and pET-28a as cloning vector.

BACKGROUND: Due to its high expression capability, recombination of Escherichia coli and pET vector has become the bioengineering preferred expression system. Because ß-lactamases mediate bacterial antimicrobial resistance, these enzymes have a substantial clinical impact. Using the E. coli expression system, several kinds of ß-lactamases have been produced. However, previous studies have been focused on characterizing target ß-lactamases, and the effects of cultivation and induction conditions on the expression efficiency of target enzymes were not addressed. RESULTS: Using pET-28a as the cloning vector and E. coli BL21(DE3) as the expression host, this study originally elucidated the effects of IPTG concentration, culture temperature, induction time, and restriction sites on recombinant ß-lactamase expression. Moreover, the effects of the target protein length and the 6 × His-tag fusion position on enzyme purification were also explored, and consequently, this study yielded several important findings. (i) Only the signal peptide-detached recombinant ß-lactamase could exist in a soluble form. (ii) Low-temperature induction was beneficial for soluble ß-lactamase expression. (iii) The closer to the rbs the selected restriction site was, the more difficult it was to express soluble ß-lactamase. (iv) The short-chain recombinant protein and the protein with His-tag fused at its C-terminus showed high affinity to the Ni2+ column. CONCLUSIONS: Based on our findings, researchers can easily design an effective program for the high production of soluble recombinant ß-lactamases to facilitate other related studies.

Magnetic Molecularly Imprinted Polymers for the Rapid and Selective Extraction and Detection of Methotrexatein Serum by HPLC-UV Analysis.

In this work, novel selective recognition materials, namely magnetic molecularly imprinted polymers (MMIPs), were prepared. The recognition materials were used as pretreatment materials for magnetic molecularly imprinted solid-phase extraction (MSPE) to achieve the efficient adsorption, selective recognition, and rapid magnetic separation of methotrexate (MTX) in the patients' plasma. This method was combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) to achieve accurate and rapid detection of the plasma MTX concentration, providing a new method for the clinical detection and monitoring of the MTX concentration. The MMIPs for the selective adsorption of MTX were prepared by the sol-gel method. The materials were characterized by transmission electron microscopy, Fourier transform-infrared spectrometry, X-ray diffractometry, and X-ray photoelectron spectrometry. The MTX adsorption properties of the MMIPs were evaluated using static, dynamic, and selective adsorption experiments. On this basis, the extraction conditions were optimized systematically. The adsorption capacity of MMIPs for MTX was 39.56 mgg-1, the imprinting factor was 9.40, and the adsorption equilibrium time was 60 min. The optimal extraction conditions were as follows: the amount of MMIP was 100 mg, the loading time was 120 min, the leachate was 8:2 (v/v) water-methanol, the eluent was 4:1 (v/v) methanol-acetic acid, and the elution time was 60 min. MTX was linear in the range of 0.00005-0.25 mg mL-1, and the detection limit was 12.51 ng mL-1. The accuracy of the MSPE-HPLC-UV method for MTX detection was excellent, and the result was consistent with that of a drug concentration analyzer.

A rapid and highly effective approach for SARS-CoV-2 nucleic acid daily testing in more than four thousand single-tube samples.

BACKGROUND: Presently, the global spread of COVID-19 is still going on, with more than 0.6 million new cases confirmed per day (as of November 20, 2021). However, since China entered a post-epidemic phase in mid-March 2020, the daily number of new domestic infections in the Chinese mainland has been maintained at almost zero or single digits, which was attributed to a series of effective measures for COVID-19 prevention and control adopted by the Chinese government. Among these measures, SARS-CoV-2 nucleic acid testing holds key role for the timely confirmation and isolation of the infections to prevent further transmission. METHODS: Referring to the national policy requirements, since April 30, 2020, The Affiliated Hospital of Qingdao University has conducted SARS-CoV-2 nucleic acid testing in its PCR laboratory for patients and social workers, as well as for environmental monitoring and employee screening. As of mid-November 2020, the daily amount of single-tube samples for nucleic acid testing rose above 4,000. RESULTS: In this article, a rapid and highly effective approach for SARS-CoV-2 nucleic acid daily testing is presented, allowing five technicians to complete nucleic acid testing in 6,500 single-tube samples in one day with a high level of quality. Using this approach, since the samples entered the PCR laboratory, all testing results were reported in 2.5-3 h with satisfactory quality control and precise reporting criterion as prerequisites. CONCLUSION: This testing approach provides a referable workflow for other testing institutions and is expected to play an important role in COVID-19 prevention and control.

Development of a standardized Gram stain procedure for bacteria and inflammatory cells using an automated staining instrument.

Gram stain is a subjective and poorly controlled test, and the resultant errors often perplex laboratory scientists. To reduce errors and make Gram stain a precisely controllable and meritorious test, a standardized Gram stain procedure for bacteria and inflammatory cells was developed using an automated staining instrument in this study. Freshly expectorated sputum specimens, used as the optimized targets, were smeared on slides by laboratory technicians, defining each slide loaded with uniform matrix and monolayer cell. And then, the staining and decolorizing time, as well as the stain and decolorant volume, were optimized as 15, 105, 1, and 25 s and 1.1, 1.4, 0.3, and 0.7 ml, respectively. Culture-positive blood specimens and original purulent fluids were used for confirming the developed standardized Gram stain procedure. Distinct tinctures of bacteria and inflammatory cells adhered to slide uniformly in a monolayer were observed, and the obtained staining results of these samples were highly consistent with their cultured results. Furthermore, according to the staining results under different staining conditions, an updated molecular mechanism of Gram stain for bacteria and the probable staining mechanism for inflammatory cells were also proposed in this study.

Bioaccumulation of tetrabromobisphenol A in a laboratory-based fish-water system based on selective magnetic molecularly imprinted solid-phase extraction.

Recently, magnetic solid-phase extraction (MSPE) using magnetic molecularly imprinted polymers (MMIPs), which is a simple process with excellent selectivity, has attracted much attention for the determination of environmental pollutants. In this study, MMIPs were used as an adsorbent to establish a selective MSPE method coupled with high-performance liquid chromatography using ultraviolet detection (HPLC-UV) for the determination of tetrabromobisphenol A (TBBPA) levels in water and fish samples. The samples were collected from a laboratory-based fish-water system after 0, 2, 5, 8, 11, 20, 30, and 50 days. We found that the concentrations of TBBPA in the sample group spiked with TBBPA decreased in the water samples over time and increased in the fish samples from 2 to 30 days, then finally decreased. The calculated bioconcentration factor (BCF) increased over time, reaching 33.98 L/kg after 50 days exposure to TBBPA. Linear and exponential kinetic models were applied to fit the correlation between BCF and exposure time, and the constant of the time-dependent BCF (Ku) ranged from 0.0364 to 1.5250 L/kg per day with a corresponding R2 of 0.6786 to 0.9985. Simplified mathematical models to evaluate the transfer characteristics of TBBPA in a laboratory-based fish-water system have been developed.

Occurrence investigation of perfluorinated compounds in surface water from East Lake (Wuhan, China) upon rapid and selective magnetic solid-phase extraction.

Using a novel magnetic nanocomposite as adsorbent, a convenient and effective magnetic solid-phase extraction (MSPE) procedure was established for selective separation and concentration of nine perfluorinated compounds (PFCs) in surface water sample. Then an ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system was employed for detection of PFCs. Good linearity of the developed analytical method was in the range of 0.5-100 ng L-1 with R2 > 0.9917, and the limits of detection (LODs) ranged from 0.029 to 0.099 ng L-1. At three fortified concentrations of 0.5, 5 and 50 ng L-1, the spiked recoveries of PFCs were in the range of 90.05-106.67% with RSDs < 12.62% (n = 3). The proposed analytical method was applied for determination of PFCs in surface water from East Lake (Wuhan, China). The total concentrations of nine PFCs ranged from 30.12 to 125.35 ng L-1, with perfluorooctane sulfonate and perfluoroctanoic acid as the most prevalent PFCs, and the greatest concentrations of PFCs were observed in Niuchao lakelet. The concentrations of the PFCs (C ≥ 11) were mostly less than the limits of quantification (LOQs), attributed to the possibility that the more hydrophobic long-chain PFCs are potential to accumulate in sediment and aquatic biota.

Fabrication of a Selective and Sensitive Sensor Based on Molecularly Imprinted Polymer/Acetylene Black for the Determination of Azithromycin in Pharmaceuticals and Biological Samples.

A new selective and sensitive sensor based on molecularly imprinted polymer/acetylene black (MIP/AB) was developed for the determination of azithromycin (AZM) in pharmaceuticals and biological samples. The MIP of AZM was synthesized by precipitation polymerization. MIP and AB were then respectively introduced as selective and sensitive elements for the preparation of MIP/AB-modified carbon paste (MIP/ABP) electrode. The performance of the obtained sensor was estimated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. Compared with non-molecularly imprinted polymer (NIP) electrodes, NIP/ABP electrodes, and MIP-modified carbon paste electrodes, MIP/ABP electrode exhibited excellent current response toward AZM. The prepared sensor also exhibited good selectivity for AZM in comparison with structurally similar compounds. The effect of electrode composition, extraction parameters, and electrolyte conditions on the current response of the sensor was investigated. Under the optimized conditions, the prepared sensor showed two dynamic linear ranges of 1.0 × 10-7 mol L-1 to 2.0 × 10-6 mol L-1 and 2.0 × 10-6 mol L-1 to 2.0 × 10-5 mol L-1, with a limit of detection of 1.1 × 10-8 mol L-1. These predominant properties ensured that the sensor exhibits excellent reliability for detecting AZM in pharmaceuticals and biological fluids without the assistance of any separation techniques. The results were validated by the high-performance liquid chromatography-tandem mass spectrometry method.

Rapid and selective extraction of multiple macrolide antibiotics in foodstuff samples based on magnetic molecularly imprinted polymers.

Magnetic molecularly imprinted polymers (MMIPs) were prepared based on surface molecular imprinting using erythromycin (ERY) as template molecule and Fe3O4 nanoparticles as support substrate. The MMIPs possessed high adsorption capacity of 94.1 mg/g for ERY and the imprinting factor was 11.9 indicating good imprinted effect for ERY. Selective evaluation demonstrated favorable selectivity of MMIPs for multiple macrolide antibiotics (MACs). Using MMIPs as adsorptive material, a rapid and convenient magnetic solid-phase extraction (MSPE) procedure was established for simultaneous and selective separation of six MACs in pork, fish and shrimp samples, then the MACs was subjected to high-performance liquid chromatography-ultraviolet (HPLC-UV) analysis. At different fortified concentrations, the extraction recoveries could reach 89.1% and the relative standard deviations were lower than 12.4%. Chromatogram revealed the response signals of MACs in spiked samples were greatly enhanced and matrix interferences were effectively eliminated after treatment with MSPE. The proposed MSPE procedure coupled with HPLC-UV realized selective and sensitive determination of multiple MACs in foodstuff samples.

Highly selective and sensitive determination of dopamine by the novel molecularly imprinted poly(nicotinamide)/CuO nanoparticles modified electrode.

A novel electrochemical sensor was proposed for the determination of dopamine (DA) based on the molecularly imprinted electropolymers (MIPs)/copper oxide (CuO) nanoparticles modified electrode. MIPs were firstly prepared by using nicotinamide as an environment-friendly monomer to selectively recognize the template molecules. CuO nanoparticles were used to enhance the number of imprinted sites per unit surface area of the electrode and then improve the selectivity and sensitivity of the electrochemical sensor. Thus, the obtained electrochemical sensor could effectively minimize the interferences caused by ascorbic acid (AA), uric acid (UA) and sample matrix. The linear range for the detection of DA was changed from 0.02 µmol L(-1) to 25 µmol L(-1) with the detection limit of 8 nmol L(-1) (S/N=3), which was lower than those of the reported MIPs-based sensor. Finally, the proposed method was applied to measure dopamine in serum samples. The spiked recoveries were changed from 96.9% to 105.9% and the RSD was not higher than 8.8%. It was shown that the proposed sensor exhibited significant promise as a reliable technique for the detection of DA in human serum samples.

Molecularly imprinted spin column extraction coupled with high-performance liquid chromatography for the selective and simple determination of trace nitrophenols in water samples.

In this study, we developed a simple and selective spin column extraction technology utilizing hydrophilic molecularly imprinted polymers as the sorbents for extracting nitrophenol pollutants in water samples (the East Lake, the Yangtze River, and wastewater). The whole procedure was achieved by centrifugation of the spin column, and multiple samples were simultaneously processed with a low volume of solvent and without evaporation. Under the optimized condition, recoveries of nitrophenol compounds on the spin column packed with hydrophilic molecularly imprinted polymers ranged from 87.3 to 92.9% and an excellent purification effect was obtained. Compared with activated carbon, multi-walled carbon nanotubes, LC-C18 sorbents, hydrophilic molecularly imprinted polymers exhibited a highly selective recognition ability for nitrophenol compounds and satisfactory sample extraction efficiency. Subsequently, the spin column extraction coupled with high-performance liquid chromatography was established, which was found to be linear in the range of 2-1000 ng/mL for 2,4-dinitropehnol and 2-nitrophenol, and 6-1000 ng/mL for 4-nitrophenol with correlation coefficients greater than 0.998. The detection limits ranged from 0.3-0.5 ng/mL. It is shown that the proposed method can be used for the determination of trace nitrophenol pollutants in complex samples, which is not only beneficial for water quality analysis but also for environmental risk assessment.

A simple and sensitive immunoassay for the determination of human chorionic gonadotropin by graphene-based chemiluminescence resonance energy transfer.

In this study, we report a strategy of chemiluminescence resonance energy transfer (CRET) using graphene as an efficient long-range energy acceptor. Magnetic nanoparticles were also used in CRET for simple magnetic separation and immobilization of horseradish peroxidase (HRP)-labeled anti-HCG antibody. In the design of CRET system, the sandwich-type immunocomplex was formed between human chorionic gonadotropin (HCG, antigen) and two different antibodies bridged the magnetic nanoparticles and graphene (acceptors), which led to the occurrence of CRET from chemiluminescence light source to graphene. After optimizing the experimental conditions, the quenching of chemiluminescence signal depended linearly on the concentration of HCG in the range of 0.1 mIU mL(-1)-10 mIU mL(-1) and the detection limit was 0.06 mIU mL(-1). The proposed method was successfully applied for the determination of HCG levels in saliva and serum samples, and the results were in good agreement with the plate ELISA with colorimetric detection. It could also be developed for detection of other antigen-antibody immune complexes by using the corresponding antigens and respective antibodies.

Highly effective removal of 2,4-dinitrophenolic from surface water and wastewater samples using hydrophilic molecularly imprinted polymers.

Novel hydrophilic molecularly imprinted polymers (MIPs) with high adsorption capacity were used as the sorbents to remove 2,4-dinitrophenol (2,4-DNP) from surface water and wastewater samples. Kinetic studies, dynamic adsorption and selectivity experiments of hydrophilic MIPs were investigated in this study. The results indicated that the maximum adsorption capacity of 2,4-DNP on hydrophilic MIPs was 138.9 mg g(-1) and kinetic experimental data were described by the pseudo-second-order model. Furthermore, the effects of flow rate, initial concentration, pH value, and humic acid on the removal efficiency of 2,4-DNP were optimized. Compared with the active carbon, carbon nanotube, C18 sorbents and common MIPs, the removal efficiency of hydrophilic MIPs (100 mg) was very high with all above 92 % even though the sampling volume was more than 1 L. Investigated results of five times adsorption-desorption cycles indicated hydrophilic MIPs were high stability. In a word, the obtained results demonstrated that hydrophilic MIPs could be used as the effective sorbents for 2,4-DNP removal in practical application.

[Determination of erythromycin residue in pork samples using molecularly imprinted solid phase extraction coupled with high performance liquid chromatography].

Molecularly imprinted polymers (MIPs) for erythromycin (ERY) were prepared by precipitation polymerization, using ERY as template molecule, methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as cross-linker and methanol/acetonitrile (1:4, v/v) as porogens. The characterization of the prepared MIPs and non-imprinted polymers (NIPs) were evaluated by scanning electron microscope (SEM) and equilibrium adsorption experiment. The results showed that the MIPs possessed the specific adsorptivity toward ERY. Scatchard analysis revealed that the apparent maximum binding capacity (Qmax) and the dissociation content (Kd) to ERY were as follows: Qmax1 = 45.24 mg/g, Kd1 = 0.028 g/L; Qmax2 = 87.53 mg/g, Kd2 = 0.20 g/L. Using the prepared MIPs as selective adsorbent, the proposed molecularly imprinted solid phase extraction (MISPE) coupled with high performance liquid chromatography (HPLC) was successfully applied to determine ERY residue in pork samples. A linear correlation was obtained over a range of 0.5-50 mg/L (r2 = 0.999 4), and the limit of detection was 0.2 mg/kg (S/N = 3). The spiked recoveries of ERY were 95.2%-104.2% with the relative standard deviations (RSDs) of less than 5%. The proposed method is selective, sensitive and reliable for the analysis of ERY residue in pork samples.

Determination of trace 2,4-dinitrophenol in surface water samples based on hydrophilic molecularly imprinted polymers/nickel fiber electrode.

A rapid, sensitive and selective electrochemical method was proposed for the determination of 2,4-dinitrophenol (2,4-DNP) in surface water samples, using hydrophilic molecular imprinted polymers (MIPs) as the recognition element and nickel (Ni) fiber as the catalytic element. Hydrophilic MIPs were synthesized using 2,4-DNP as the template, acrylamide as the monomer, glycidilmethacrylate as the pro-hydrophilic co-monomer and acetonitrile as the solvent. Hydrophilic modification could enhance the accessibility of 2,4-DNP to the imprinted cavities and improve the selective recognition properties of traditional MIPs in water medium. Subsequently, hydrophilic MIPs/Ni fiber electrode was prepared to determine trace 2,4-DNP by cyclic voltammetry. The parameters affecting the analytical performance were investigated. Under optimized conditions, the linear range was 0.7-30 µg L⁻¹ and the detection limit was 0.1 µg L⁻¹. Finally, the proposed method was applied to measure 2,4-DNP in surface water samples. The spiked recoveries were changed from 91.3% to 102.6% and the RSD was not higher than 5.1%. There was no statistically significant difference between the results obtained by the proposed method and the traditional chromatographic method.