Complicated Biological Samples Research Articles

Electrochemical Detection of Dopamine: Use of Covalently Bonded Ag (Silver) - Au (Gold) Bimetallic Nanoparticles

Dopamine (DA) is a crucial neurotransmitter that is generated by dopaminergic neurons in the central nervous system. We have produced and studied aspartic acid-capped gold nanoparticles and thioctic acid-capped silver nanoparticles, and we have covalently linked them via EDC-NHS coupling. The heightened stability and reproducibility of the Ag-Au-Nafion modified glassy carbon electrode (GCE) allowed for the sensitive and accurate detection of dopamine in PBS buffer at pH 6. The electrode modification approach’s selectivity and sensitivity as well as the possible interference of other compounds present in complicated biological samples, require more study. The creation of more precise and sensitive electrochemical sensors for dopamine detection is aided by this discovery, which has broad implications for medical diagnostics.

Responsive β-Diketonate-europium(III) Complex-Based Probe for Time-Gated Luminescence Detection and Imaging of Hydrogen Sulfide In Vitro and In Vivo.

As a crucial biological gasotransmitter, hydrogen sulfide (H2S) plays important roles in many pathological and physiological processes. Highly selective and sensitive detection of H2S is significant for the precise diagnosis and evaluation of diverse diseases. Nevertheless, challenges remain in view of the interference of autofluorescence in organisms and the stronger reactivity of H2S itself. Herein, we report the design and synthesis of a novel H2S-responsive β-diketonate-europium(III) complex-based probe, [Eu(DNB-Npketo)3(terpy)], for background-free time-gated luminescence (TGL) detection and imaging of H2S in autofluorescence-rich biological samples. The probe, consisting of a 2,4-dinitrobenzenesulfonyl (DNB) group coupled to a β-diketonate-europium(III) complex, shows almost no luminescence owing to the existence of intramolecular photoinduced electron transfer. The cleavage of the DNB group by a H2S-triggered reaction results in the recovery of the long-lived luminescence of the Eu3+ complex, allowing the detection of H2S in complicated biological samples to be performed in TGL mode. The probe showed a fast response, high specificity, and high sensitivity toward H2S, which enabled it to be successfully used for the quantitative TGL detection of H2S in tissue homogenates of mouse organs. Additionally, the low cytotoxicity of the probe allowed it to be further used for the TGL imaging of H2S in living cells and mice under different stimuli. All of the results suggested the potential of the probe for the investigation and diagnosis of H2S-related diseases.

Design of Ratio-Fluorescence Nanohybrid Based on Radix Hedysari Green-Synthesized CDs and GSH-AuNCs for Sensitive Detection of Cefodizime Sodium in Urine Sample.

A dual-emission ratio-fluorescent sensing nanohybrid based on Radix Hedysari green-synthesized carbon quantum dots (CDs) and glutathione-functionalized gold nanoclusters (GSH-AuNCs) had been developed for the determination of cefodizime sodium (CDZM). The designed fluorescence nanohybrid had two significant fluorescence emission peaks at 458 nm and 569 nm when excited at 360 nm, which was attributed to the CDs and GSH-AuNCs. With the addition of CDZM, the fluorescence at 458 nm was slightly weakened while the fluorescence at 569 nm was enhanced obviously. Based on the relationship between the I569/I458 fluorescence intensity ratio and the concentration of CDZM, the designed nanohybrid exhibited a good linearity range of 1.0-1000.0 μM and the limit of detection (LOD) was 0.19 μM. The method was finally applied in the detection of CDZM in urine, showing the potential applications in complicated biological samples.

Fabrication of 3D aligned porous nanocellulose-based aerogel for enrichment of the histidine-containing polypeptides

The high-throughput immobilized metal affinity chromatography (IMAC) material is urgently required to improve the efficiency of separation and purification of tagged peptides in related biomedical applications. Herein, the amino-thiourea-modified nanocellulose (S-TOCN) -polyvinyl alcohol (PVA) composite aerogel with aligned pores was prepared by a directional freeze-casting technique and then used to immobilize metal ions (M2+) for the selective separation and purification of histidine-containing peptides from protein hydrolysates. The obtained S-TOCN-PVA materials with an oriented porous structure exhibited excellent mechanical properties, good adsorption performance, and reusability under optimal conditions. The adsorption capacity of the S-TOCN-PVA aerogel for metal ions was as follows: Cu2+>Ni2+>Zn2+>Co2+, and the higher metal ion chelating amount of the S-TOCN-PVA composite aerogel for Cu2+ and Ni2+ reached 87.21 mg/g at pH=7 and 83.66 mg/g at pH=6 in the 2 h incubation period, respectively. The enrichment effects of Cu2+/S-TOCN-PVA and Ni2+/S-TOCN-PVA aerogels with the selectivity of His-containing peptides from apricot kernel protein hydrolysates were up to 3.99 folds and 4.81 folds, respectively, for one cycle. Furthermore, after 5cycles, the Ni2+/S-TOCN-PVA aerogels remained at 87.5% of their initial adsorption capacity (848 mg/g), and the concentration increased by 39.05-fold. Molecular docking simulations show that the oxygen of the carboxyl group (-C-O and -CO) and the hydrogen of the imidazole group on the His molecule are mainly bonded to the hydrogen of the amino groups and the oxygen of the carboxyl group (C-O-C) in the structure of M2+/S-TOCN-PVA aerogels. The fabricated metal ion-immobilized 3D-aligned porous nanocellulose-based aerogels are promising IMAC materials for efficient separation and purification of the histidine-containing polypeptides from complicated biological samples.

Combining EDTA-intercalated (Zn-Al) layered double hydroxides/graphite and two-phase hollow fiber for liquid phase microextraction of nonsteroidal anti-inflammatory drugs

This study synthesized and characterized layered double hydroxides (LDHs) intercalated with ethylenediaminetetraacetic acid(EDTA) to increase the basal spacing of electrosynthesized Zn-Al-LDHs on pencil graphite (PG). The characteristic verification of the Zn-Al-EDTA-LDH nanostructures was conducted utilizing Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), energy dispersive and X-ray analysis (EDS), and X-ray diffraction (XRD). To improve the extraction efficiency, Zn-Al-EDTA-LDHs as adsorbent in solid phase microextraction (SPME) fiber was combined with hollow fiber liquid–liquid phase microextraction (HF-LLPME) to investigate the extraction efficiency of HF-LLME@PG-SPME technique the chosen non-steroidal anti-inflammatory drugs (NSAIDs) from aqueous solution Several variables, comprising extraction time, desorption time, stirring rate, desorption volume, pH of donor phase, and salt effects were screened and optimized by performing placket-Burman design (PBD). After evaluating the extraction and desorption procedures under optimal conditions, NSAIDs analysis was performed employing high-performance liquid chromatography-ultraviolet detection (HPLC-UV). Compared to the most common clean-up and sample preparation methods, the current study focused on developing a greener analytical chemistry process due to reduced solvent consumption, cost-effectiveness, miniaturization and simplified design. The proposed method for NSAIDs exhibited a good linear dynamic range (LDRs) (1.00–200.00 µg/L) with a coefficient of determination of > 0.9956, low limits of detection (LODs) (0.26–0.29 µg/L) and acceptable limits of quantifications (LOQs) (0.86–0.96 µg/L). Relative standard deviations of 5.31 % could be determined. The absolute recoveries of the urine sample analyses could be obtained in a 89.91–97.61 % range and enrichment factors (EFs) (178.92 to 183.88). Investigations of the developed method were also conducted in various urine samples containing the selected drugs. As a result of the obtained recoveries, the method proved to be valuable and suitable to complicated biological samples.

Ferrocene-chalcone-based probe for the fluorescence and electrochemical dual-mode detection of carboxylesterase

Carboxylesterases (CEs) are widely distributed in the body and catalyze the hydrolysis of endogenous and exogenous substrates. They play important roles in drug metabolism, and abnormal levels often result in numerous diseases such as cancers. Therefore, it is important to establish a highly selective and sensitive probe for the real-time sensing of CEs activity. Herein, we synthesized a novel fluorescence-electrochemical multichannel probe 1 containing a ferrocene unit, which not only serves as part of the structure of probe 1 but also provides electrochemical signals. The probe exhibited high selectivity to CEs with a “Turn-On” fluorescence response and was applied for imaging in complicated biological samples including Staphylococcus aureus, living cells, and zebrafish. Furthermore, the hydrolysis of the acetoxy group of probe 1 by CEs decreased the electrochemical signal. All these results suggest that probe 1 can act as a multi-modal probe for real-time tracking of the activity CEs in complex biological environments.

An all-in-one enzymatic DNA network based on catalytic hairpin assembly for label-free and highly sensitive detection of APE1

Apurinic/apyrimidinic endonuclease 1 (APE1), identified as a prospective cancer biomarker, plays a vital role in the occurrence and progression of cancer cell lines and impacts on genome stability. However, conventional approaches typically rely on the interactions between the antigen and antibody, limiting their utility for qualitative assessments of APE1 expression. Herein, an all-in-one enzymatic DNA network (EDN) assay with catalytic hairpin assembly for label-free and ultrasensitive detection of APE1 has been developed. In this work, the blocking strand can inhibit the initiator by obstructing the complementary region, preventing the hairpin from hybridizing in the absence of APE1 targets. While the presence of targets can activate the unlocking of the initiator, which can trigger the catalytic hairpin reaction, and increase the fluorescent signal. Under optimal conditions, the developed sensing method can detect the target APE1 down to 4.78 × 10−6 U mL−1 with a wide linear range from 5 × 10−6 U mL−1 to 30 U mL−1. This strategy has also been successfully applied to the analysis of complicated biological samples compared to ELISA, demonstrating its potential applications in biochemical and molecular biology research as well as clinical diagnostics. Overall, benefiting from the high amplification efficiency, this strategy has successfully and simply detected low-abundance APE1 without additional enzyme isolation steps, presenting great potential for clinical detection applications.

A smart TESTER for reliable discrimination of cancer-derived small extracellular vesicles

Cancer-derived small extracellular vesicles (csEVs) are crucial liquid biopsy indicators that reflect the presence and progression of many malignancies. However, reliable discrimination of csEVs remains a great challenge owing to the interference from normal sEVs (nsEVs) and low abundance in the early stages of cancer. In this work, we developed a Two-Elements Selectively Triggered csEVs Recognization (TESTER) strategy for selective identification of csEVs from the complex clinical body fluid samples. This method was based on the MNAzyme-controlled synchronous recognition to EpCAM and CD63 proteins on the membrane of csEVs. Efficient recognition to csEVs via EpCAM aptamer and CD63 aptamer prompted the release of Partzyme A and Partzyme B probes to induce a MNAzyme structure formation, resulting in the cyclic cleavage of substrate chain to produce cascade fluorescence signal amplification. The detection threshold of the developed TESTER approach for csEVs in complicated biological samples was 72 particles μL−1, accomplishing the highly sensitive and selective quantification of csEVs. At the same time, we successfully constructed a new platform for bimolecular simultaneous recognition, which provides a good idea for the construction of bimolecular-activated detection switch in the future.

A butterfly-shaped ESIPT dye for pattern recognition of metal ions

Accurate monitoring of versatile metal ions in complicated environmental or biological samples is meaningful for avoiding heavy metal poisoning accidents. To reduce the production cost of a metal ion sensor array, we intended to develop solvent directed fluorescent sensor arrays on the basis of a single dye molecule. Herein, two fluorescent ESIPT dyes named SC1 and SC2 were designed by incorporating different numbers of benzothiazole units as the metal ion binding site. It was discovered that SC1 was highly selective toward Zn2+ in all tested solvents, while a solvent directed cross-responsive sensing behavior was observed for SC2. Therefore a three-component sensor array SC2SA was readily constructed by simply dissolving SC2 in common solvents including ethanol, DMF and DMSO. The sensor array was determined to be accurate for semi-quantitative discrimination and detection of Zn2+, Cd2+, Mg2+, Fe2+ and Fe3+ in diluted serum media. Moreover, sensor array SC2SA was also successfully applied for differentiating Zn2+/Cd2+ and Fe2+/Fe3+ in pure and mixed forms and reliable identification of unknown Zn2+, Cd2+ and Mg2+ in diluted serum samples.

Specific capture and determination of glycoprotein using a hybrid epitopes and monomers-mediated molecular-imprinted polymer enzyme-free electrochemical biosensor

A novel molecular-imprinted polymer (MIP)-based enzyme-free biosensor was created for the selective detection of glycoprotein transferrin (Trf). For this purpose, MIP-based biosensor for Trf was prepared by electrochemical co-polymerization of novel hybrid monomers 3-aminophenylboronic acid (M-APBA) and pyrrole on aglassy carbon electrode (GCE) modified with carboxylated multi-walled carbon nanotubes (cMWCNTs). Hybrid epitopes of Trf (C-terminal fragment and glycan) have been selected as templates. The produced sensor exhibited great selective recognition ability toward Trf under optimal preparation conditions, offering good analytical range (0.125-1.25μM) with a detection limit of 0.024μM. Theproposed hybrid epitope in combination with hybrid monomer-mediated imprinting strategy was successfully applied to detect Trf in spiked human serum samples, with recoveries and relative standard deviations ranging from 94.7 to 106.0% and 2.64 to 5.32%, respectively. This study provided a reliable protocol for preparing hybrid epitopes and monomers-mediated MIP for the synergistic and effective determination of glycoprotein in complicated biological samples.

Interference reduction isothermal nucleic acid amplification strategy for COVID-19 variant detection

Common reference methods for COVID-19 variant diagnosis include viral sequencing and PCR-based methods. However, sequencing is tedious, expensive, and time-consuming, while PCR-based methods have high risk of insensitive detection in variant-prone regions and are susceptible to potential background signal interference in biological samples. Here, we report a loop-mediated interference reduction isothermal nucleic acid amplification (LM-IR-INA) strategy for highly sensitive single-base mutation detection in viral variants. This strategy exploits the advantages of nicking endonuclease-mediated isothermal amplification, luminescent iridium(III) probes, and time-resolved emission spectroscopy (TRES). Using the LM-IR-INA strategy, we established a luminescence platform for diagnosing COVID-19 D796Y single-base substitution detection with a detection limit of 2.01 × 105 copies/μL in a linear range of 6.01 × 105 to 3.76 × 108 copies/μL and an excellent specificity with a variant/wild-type ratio of significantly less than 0.0625%. The developed TRES-based method was also successfully applied to detect D796Y single-base substitution sequence in complicated biological samples, including throat and blood, and was a superior to steady-state technique. LM-IR-INA was also demonstrated for detecting the single-base substitution D614G as well as the multiple-base mutation H69/V70del without mutual interference, indicating that this approach has the potential to be used as a universal viral variant detection strategy.

A Background-Free SERS Strategy for Sensitive Detection of Hydrogen Peroxide.

The accurate and sensitive detection of biomolecules by surface-enhanced Raman spectroscopy (SERS) is possible, but remains challenging due to the interference from biomolecules in complex samples. Herein, a new SERS sensor is developed for background-free detection of hydrogen peroxide (H2O2) with an ultralow detection limit (1 × 10-10 mol/L), using a Raman-silent strategy. The Au microparticles (Au-RSMPs) resembling rose-stones are devised as SERS substrates with a high enhancement effect, and 4-mercaptophenylboronic acid (4-MPBA) is selected as an H2O2-responsive Raman reporter. Upon the reaction with H2O2, the phenylboronic group of 4-MPBA was converted to a phenol group, which subsequently reacted with 4-diazonium-phenylalkyne (4-DP), an alkyne-carrying molecule via the azo reaction. The formed product exhibits an intense and sharp SERS signal in the Raman-silent region, avoiding interference of impurities and biomolecules. As a proof-of-concept demonstration, we show that this SERS sensor possesses significant merits towards the determination of H2O2 in terms of broad linear range, low limit of detection, and high selectivity, showing promise for the quantitative analysis of H2O2 in complicated biological samples.

Combination of in-situ electro synthesized Zn–Al-LDH@ pencil graphite fiber and three phase hollow fiber LPME for microextraction of some antibiotics in urine samples and quantification via HPLC-UV

In the present study, a combination of two useful extraction techniques including fiber solid phase microextraction based on the new synthesized sorbent and the porous hollow fiber liquid phase microextraction is reported. The selective anion-exchangeable Zn–Al-layered double hydroxide as a fiber was electrodeposited on the surface of pencil graphite substrate through simple and cost-effective method. The prepared fiber was characterized and put into the lumen of the porous hollow fiber, which was filled by alkaline solution. The obtained optimum condition for the method was as follows: donor phase HCl concentration of 0.10 mM, acceptor phase NaOH concentration of 7.60 mM and extraction time of 16 min. It is necessary to adjust the concentration of the donor phase in order to extract acidic analytes from an aqueous sample into an organic phase. The method was used for the quantification of three antibiotics including amoxicillin, ciprofloxacin and cefixime. The extracted antibiotics were analyzed by high performance liquid chromatography-ultraviolet detection (HPLC-UV). In the optimal conditions, the linearity of the proposed method was in the range of 0.50–500.00 μg L−1 for selected analytes in water and urine matrices. All calibration curves showed determination of coefficient higher than 0.9958. The limits of detection and limits of quantification were calculated to be between 0.08 and 0.16 μg L−1 and 0.28–0.56 μg L−1, respectively. In order to define the precision of the method, the inter-day and intra-day relative standard deviations were determined between 3.25 and 5.73% in the three selected concentrations. The enrichment factors (EFs) were between 46.20 and 55.81 for selected drugs. Moreover, the calculated absolute recoveries were between 69.30 and 83.71%. The relative recoveries were obtained in the range of 93.00–105.00%. The proposed method was also employed for analysis of various urine samples containing the selected drugs. The obtained recoveries indicated that the method was useful and applicable in complicated biological samples.

Intensity and phase imaging through scattering media via deep despeckle complex neural networks

The existence of a scattering medium causes the degeneration of intensity and phase information, especially in biological imaging. The present techniques to address this challenge only focus on the reconstruction of intensity information, yet few attempts have tried to recover the phase information. We propose a method to simultaneously predict both intensity and phase information from a speckle image employing a deep despeckle complex neural network (DespeckleNet). By combining the advantages of both the complex network and the generative adversarial network framework, our method enables the high contrast single-shot imaging of complicated biological samples through scattering media without labeling. Various experiments demonstrate the superior reconstruction and generalization performance of our method under multiple types of biological samples with different scattering media. We also provide the real-time observation of living cellular activities without any contaminations or damages to the cells. Our method offers simple yet effective imaging through scattering media and paves the way for real-time unlabeled biological imaging.

Fabrication of copolymer brushes grafted superporous agarose gels: Towards the ultimate ideal particles for efficient affinity chromatography

A composite immobilized-metal affinity agarose particle was designed for the selective separation and purification of histidine-tagged proteins from complicated biological samples. The composite particle was constructed using superporous agarose particles as supporting matrix, flexible copolymer brushes as scaffolds to render higher ligand densities, and Ni2+-chelated iminodiacetic acids as recognition elements. Superporous agarose composite particles endow high permeability and interfering substance tolerance. The copolymer brush was prepared by surface-initiated atom transfer radical polymerization of N-isopropylacrylamide and glycidyl methacrylate, followed by iminodiacetic acids and Ni2+ ions. The physical and chemical properities of the composite particle were thoroughly investigated. The composite particles were shown to be able to selectively separate histidine-tagged recombinant proteins in the presence of high quantities of interfering chemicals in a model protein-binding experiment. By altering the temperature, the protein binding of the composite particles can be modulated. The superporous agarose particles supported polymer brush enables fast and efficient separation and purification of target proteins with high permeability, low backpressure, and high interfering matrix tolerance, which pave the path for bioseparation through designing and fabrication of novel agarose particles-based functional materials.

One-step fabrication of nitrogen-rich linear porous organic polymer-based micron-sized sphere for selective enrichment of glycopeptides

In this work, nitrogen-rich linear porous organic polymers (POPs) were designed to improve glycopeptides enrichment efficiency in HILIC. Two kinds of novel POPs (CH-pPAL and CH-mPAL) were one-step prepared via the Schiff-base condensation reaction and intermolecular hydrogen-bonded using a flexible amine monomer (carbohydrazide, CH) to react with two aldehyde monomers, p-phthalaldehyde (pPAL) and m-phthalaldehyde (mPAL), respectively. The specific surface area of CH-pPAL and CH-mPAL was 238 and 232 m2 g−1, respectively. Based on the hydrogen-bond interaction, several narrow peaks existed in the PXRD patterns. Particularly, micron-sized spherical CH-pPAL possessed a honeycomb-like orderly tunnel structure. The contact angle of CH-pPAL and CH-mPAL severally was 35.4° and 28.8°, respectively, indicating excellent hydrophilicity of as-synthesized POPs. They both demonstrated high sensitivity and high adsorption capacity in glycopeptides enrichment. The 851 and 794 unique glycopeptides derived from 170 and 200 N-glycosylated proteins were identified after the enrichment by CH-pPAL and CH-mPAL within three independent replicates of proteins extracted from human serum, respectively. Therefore, these CH-based hydrophilic POP materials would be applied in the enrichment and identification of low-abundance N-linked glycopeptides from complicated biological samples.

Metal-organic framework-mediated synthesis of hierarchical layered double hydroxide for high-efficiency enrichment of phosphopeptides

When applied as adsorbents for phosphopeptides enrichment, two-dimensional (2D) layered double hydroxides (LDHs) are usually limited by the disadvantages of buried affinity sites and reduced specific surface area. Multifarious exfoliation strategies have been implemented to compensate for these deficiencies, but tedious exfoliation process cannot meet the requirements of LDHs as high-efficiency adsorbents. Incorporating LDHs with three-dimensional (3D) template can avoid tedious exfoliation and produce hierarchical LDHs with large specific surface area and massive affinity sites. Herein, a hierarchical LDH (denoted as Fe3O4@ZIF-8@Zn–Ga LDH) was prepared by metal-organic framework (MOF)-mediated synthesis strategy, and a magnetic solid-phase extraction (MSPE) platform was constructed and employed for phosphopeptides enrichment with high efficiency. The unique 3D structure and abundant metal nodes of MOF provide 3D template and metal sources for in-situ nucleation and generation of LDH. Large specific surface area and massive exposed Zn and Ga endow Fe3O4@ZIF-8@Zn–Ga LDH with high enrichment efficiency toward phosphopeptides from complicated biological samples. With the aid of mass spectrometry (MS) techniques, we profiled endogenous or global phosphopeptides from human saliva and serum, which proved the practical application value of this material.

Sensitive detection of exosomes by gold nanoparticles labeling inductively coupled plasma mass spectrometry based on cholesterol recognition and rolling circle amplification

Exosomes, potential biomarkers for liquid biopsy, provide a promising opportunity for early cancer diagnosis in a noninvasive manner. However, direct analysis of exosomes in complicated biological samples is still challenging. Herein, a cholesterol recognition assay was proposed for detection of exosomes by gold nanoparticles (Au NPs) labeling inductively coupled plasma mass spectrometry. Specifically, exosomes were captured by anti-CD63, and then cholesterol-modified rolling circle amplification product was inserted into the membrane of exosomes for signal amplification. Simultaneously, Au NPs-labelled DNA was prepared for labeling and read-out signal. The proposed method achieved the quantification of the exosome concentration in a range of 10-106 particles/μL with a limit of detection of 1.5 particles/μL. The method was applied for the direct detection of exosomes in human serum in a purification-free way, and the analytical result coincided with that obtained by nanoparticle tracking analysis. Real sample analysis manifested that the exosome concentration in the serum of oral squamous cell carcinoma patients was higher than that in the healthy volunteers’ serum. The developed method is sensitive, selective, accurate and can be used for the direct detection of exosomes in human serum, providing a new opportunity for exosome-based liquid biopsy in early cancer diagnosis.

Multi-extraction system with identical supported semi-liquid membrane: Enhanced stability for coextraction of acidic and basic drugs

Coextraction of different groups of analytes is vital for saving sample volumes and simplifying analytical procedures in bioanalysis. Conventionally, coextraction was achieved by using multi-extraction systems with different supported liquid membranes (SLMs). However, the different membrane solvents tended to diffuse into the aqueous solutions and the other SLM to reach distribution equilibrium during extraction process, causing the system instability. In this work, a stable multi-extraction system (integration of liquid-phase microextraction and electromembrane extraction, LPME/EME) based on the identical supported semi-liquid membrane (SsLM) was developed. Principally, the state of distribution equilibrium of the membrane solvent (polypropylene glycol with molecular weight 4000) in SsLM could be reached at the beginning of extraction, which enhanced the coextraction stability. With this multi-extraction system, acidic and basic analytes were simultaneously extracted from practical biological samples. The extraction recoveries of the six model drugs in undiluted urine samples were over 70%. Followed by LC-MS/MS, the limits of quantification (LOQs) were in the range of 5–10 ng mL−1. The multi-extraction system using the identical SsLM in this study shows promising potential in construction of other stable multi-extraction systems (e.g., LPME/LPME and EME/EME) in the future, which will greatly benefit the group separation of analytes in complicated biological samples.

Ultrasensitive Uracil-DNA Glycosylase Activity Assay and Its Inhibitor Screening Based on Primer Remodeling Jointly via Repair Enzyme and Polymerase.

The development of isothermal nucleic acid amplification techniques has great significance for highly sensitive biosensing in modern biology and biomedicine. A facile and robust exponential rolling circle amplification (RCA) strategy is proposed based on primer-remodeling amplification jointly via a repair enzyme and polymerase, and uracil-DNA glycosylase (UDG) is selected as a model analyte. Two kinds of complexes, complex I and complex II, are preprepared by hybridizing a circular template (CT) with a uracil-containing hairpin probe and tetrahydrofuran abasic site mimic (AP site)-embedded fluorescence-quenched probe (AFP), respectively. The target UDG specifically binds to complex I, resulting in the generation of an AP site, followed by cleavage via endonuclease IV (Endo IV) and the successive trimming of unmatched 3' terminus via phi29 DNA polymerase, thus producing a useable primer-CT complex that actuates the primary RCA. Then, numerous complex II anneal with the first-generation RCA product (RP), generating a complex II-RP assembly containing AP sites within the DNA duplex. With the aid of Endo IV and phi29, AFP, as a pre-primer in complex II, is converted into a mature primer to initiate additional rounds of RCA. So, countless AFPs are cleaved, releasing remarkably strong fluorescent signals. The biosensor is demonstrated to enable rapid and accurate detection of the UDG activity with an improved detection limit as low as 4.7 × 10-5 U·mL-1. Moreover, this biosensor is successfully applied for UDG inhibitor screening and complicated biological samples analysis. Compared to the previous exponential RCA methods, our proposed strategy offers additional advantages, including excellent stability, optional design of CT, and simplified operating steps. Therefore, this proposed strategy may create a useful and practical platform for ultrasensitive detection of low levels of analytes in clinical diagnosis and fundamental biomedicine research.