Separation/Concentration‐signal‐amplification in‐One Method Based on Electrochemical Conversion of Magnetic Nanoparticles for Electrochemical Biosensing

We propose a separation/concentration‐signal‐amplification in‐one method based on electrochemical conversion (ECC) of magnetic nanoparticles (MNPs) to develop a facile and sensitive electrochemical biosensor for chloramphenicol (CAP) detection. Briefly, aptamer‐modified magnetic nanoparticles (MNPs‐Apt) was designed to capture CAP in sample, then the MNPs‐Apt composite was conjugated to Au electrode through the DNA hybridization between the unoccupied aptamer and a strand of complementary DNA. The ECC method was applied to transfer MNPs labels to electrochemically active Prussian blue (PB). The anodic and cathodic currents of PB were taken for signal readout. Comparing with conventional methods that require electrochemically active labels and related sophisticated labelling procedures, this method explored and integrated the magnetic and electrochemical properties of MNPs into one system, in turn realized magnetic capturing of CAP and signal generation without any additional conventional labels. Taking advantages of the high abundance of iron content in MNPs and the refreshing effect deriving from ECC process, the method significantly promoted the signal amplification. Therefore, the proposed biosensors exhibited linear detection range from 1 to 1000 ng mL⁻¹ and a limit of detection down to 1 ng mL⁻¹, which was better than or comparable with those of most analogues, as well as satisfactory specificity, storage stability and feasibility for real samples. The developed method may lead to new concept for rapid and facile biosensing in food safety, clinic diagnose/therapy and environmental monitoring fields.     

Fe3O4 nanoparticles-enhanced SPR sensing for ultrasensitive sandwich bio-assay

Magnetic nanoparticles (MNPs) have been receiving increasing attention because of its great potentials in bioseparation. However, the separation products are difficult to be detected by general method due to their extremely small size. Here, we demonstrate that MNPs can greatly enhance the signal of surface plasmon resonance spectroscopy (SPR). Features of MNPs-aptamer conjugates as a powerful amplification reagent for ultrasensitive immunoassay are reported in this work for the first time. In order to evaluate the sensing ability of MNPs-aptamer conjugates as an amplification reagent, a sandwich SPR sensor is constructed by using thrombin as model analyte. Thrombin, captured by immobilized anti-thrombin aptamer on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 0.017 nM after MNPs-aptamer conjugates is used as amplification reagent. At the same time, the excellent selectivity of the present biosensor is also confirmed by using three kinds of proteins (BSA, human IgM and human IgE) as controls. These results confirm that MNPs is a powerful sandwich element and an excellent amplification reagent for SPR based sandwich immunoassay and SPR has a great potential for the detection of MNPs-based bioseparation products.     

Precise characterization method of antibody‐conjugated magnetic nanoparticles for pathogen detection using stuffer‐free multiplex ligation‐dependent probe amplification

Antibody‐conjugated magnetic nanoparticles (Ab‐MNPs) have potential in pathogen detection because they allow target cells to be easily separated from complex sample matrices. However, the sensitivity and specificity of pathogen capture by Ab‐MNPs generally vary according to the types of MNPs, antibodies, and sample matrices, as well as preparation methods, including immobilization. Therefore, achieving a reproducible analysis utilizing Ab‐MNPs as a pathogen detection method requires accurate characterization of Ab‐MNP capture ability and standardization of all handling processes. In this study, we used high‐resolution CE‐single strand conformational polymorphism coupled with a stuffer‐free multiplex ligation‐dependent probe amplification system to characterize Ab‐MNPs. The capture ability of Ab‐MNPs targeting Salmonella enteritidis and nine pathogens, including S. enteritidis, was analyzed in phosphate buffer and milk. The effect of storage conditions on the stability of Ab‐MNPs was also assessed. The results showed that the stuffer‐free multiplex ligation‐dependent probe amplification system has the potential to serve as a standard characterization method for Ab‐MNPs. Moreover, the precise characterization of Ab‐MNPs facilitated robust pathogen detection in various applications.     

A novel cartridge for nucleic acid extraction,amplification and detection of infectious disease pathogens with the help of magnetic nanoparticles

Nucleic acid detection (NAD) based on real-time polymerase chain reaction (real-time PCR) is gold standard for infectious disease detection. Magnetic nanoparticles (MNPs) are widely used for nucleic acid extraction (NAE) because of their excellent properties. Microfluidic technology makes automated NAD possible. However, most of the NAD microfluidic chips are too complex to be applied to point-of-care (POC) testing. In this paper, a simple-structure cartridge was developed for POC detection of infectious diseases. This self-contained cartridge can be divided into a magnetic-controlled NAE part, a valve-piston combined fluidic control part and a PCR chip, which is able to extract nucleic acid from up to 500 µL of liquid samples by MNPs and finish the detection process from “sample in” to “answer out” automatically. Performance tests of the cartridges show that it met the demands of automated NAD. Results of on-cartridge detection of hepatitis B virus (HBV) demonstrated that this system has good uniformity and no cross-contamination between different cartridges, and the limit of detection (LOD) of this system for HBV in serum is 50 IU/mL. Multiplex detections of severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2) with a concentration of 500 copies/mL were carried out on the system and 100% positive detection rate was achieved.     

Fluorogenic Ag+–Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining

Silver staining, which exploits the special bioaffinity and the chromogenic reduction of silver ions, is an indispensable visualization method in biology. It is a most popular method for in‐gel protein detection. However, it is limited by run‐to‐run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) analysis; limitations that are largely attributed to the tricky chromogenic visualization. Herein, we reported a novel water‐soluble fluorogenic Ag⁺ probe, the sensing mechanism of which is based on an aggregation‐induced emission (AIE) process driven by tetrazolate‐Ag⁺ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains.     

Subnanomolar Detection of Oligonucleotides through Templated Fluorogenic Reaction in Hydrogels: Controlling Diffusion to Improve Sensitivity

Oligonucleotide‐templated reactions are valuable tools for nucleic acid sensing both in vitro and in vivo. They are typically carried out under conditions that make any reaction in the absence of template highly unfavorable (most commonly by using a low concentration of reactants), which has a negative impact on the detection sensitivity. Herein, we report a novel platform for fluorogenic oligonucleotide‐templated reactions between peptide nucleic acid probes embedded within permeable agarose and alginate hydrogels. We demonstrate that under conditions of restricted mobility (that is, limited diffusion), non‐specific interactions between probes are prevented, thus leading to lower background signals. When applied to nucleic acid sensing, this accounts for a significant increase in sensitivity (that is, lower limit of detection). Optical nucleic acid sensors based on fluorogenic peptide nucleic acid probes embedded in permeable, physically crosslinked, alginate beads were also engineered and proved capable of detecting DNA concentrations as low as 100 pm .     

Magnetic nanoparticles for the manipulation of proteins and cells

In the rapidly developing areas of nanobiotechnology, magnetic nanoparticles (MNPs) are one type of the most well-established nanomaterials because of their biocompatibility and the potential applications as alternative contrast enhancing agents for magnetic resonance imaging (MRI). While the development of MNPs as alternative contrast agents for MRI application has moved quickly to testing in animal models and clinical trials, other applications of biofunctional MNPs have been explored extensively at the stage of qualitative or conceptual demonstration. In this critical review, we summarize the development of two straightforward applications of biofunctional MNPs--manipulating proteins and manipulating cells--in the last five years or so and hope to provide a relatively comprehensive assessment that may help the future developments. Specifically, we start with the examination of the strategy for the surface functionalization of MNPs because the applications of MNPs essentially depend on the molecular interactions between the functional molecules on the MNPs and the intended biological targets. Then, we discuss the use of MNPs for manipulating proteins since protein interactions are critical for biological functions. Afterwards, we evaluate the development of the use of MNPs to manipulate cells because the response of MNPs to a magnetic field offers a unique way to modulate cellular behavior in a non-contact or "remote" mode (i.e. the magnet exerts force on the cells without direct contact). Finally, we provide a perspective on the future directions and challenges in the development of MNPs for these two applications. By reviewing the examples of the design and applications of biofunctional MNPs, we hope that this article will provide a reference point for the future development of MNPs that address the present challenges and lead to new opportunities in nanomedicine and nanobiotechnology (137 references).     

G-Quadruplex-based DNAzyme for colorimetric detection of cocaine: using magnetic nanoparticles as the separation and amplification element

The appearance of the aptamer provides good recognition elements for small molecules, especially for drugs. In this work, by combining the advantages of magnetic nanoparticles (MNPs) with colorimetric drug detection using hemin-G-quadruplex complex as the sensing element, we report a simple and sensitive DNAzyme-based colorimetric sensor for cocaine detection in a 3,3,5,5-tetramethylbenzidine sulfate (TMB)-H(2)O(2) reaction system. The whole experimental processes are simplified. Cocaine aptamer fragments, SH-C2, are covalently labeled onto the amine-functionalized MNPs. When the target cocaine and another cocaine aptamer fragments (C1) grafted with G-riched strand AG4 (i.e. C1-AG4) are present simultaneously, the C2 layer on MNPs hybridizes partly with C1-AG4 to bind the cocaine. The C1-AG4 can be combinded with hemin to form DNAzyme which can effectively catalyze the H(2)O(2)-mediated oxidation of TMB, giving rise to a change in solution color. Importantly, using MNPs as the separation and amplification elements could effectively reduce the background signal and the interference from the real samples. A linear response from 0.1 μM to 20 μM is obtained for cocaine and a detection limit of 50 nM is achieved, which provides high sensitivity and selectivity to detect cocaine.     

Magnetically confined hydrophobic nanoparticles for the microextraction of endocrine-disrupting phenols from environmental waters

In this article, a solid-phase extraction approach, which takes advantage of the good extraction capabilities of hydrophobic magnetic nanoparticles (MNPs), is presented. The new approach involves the deposition of a thin layer of MNPs in a dedicated stirring unit based on the dual function of a mini-magnet. The system allows the extraction of the analytes in a simple and efficient way. The approach, which reduces the negative effect of the aggregation tendency of hydrophobic MNPs, is characterized for the resolution of a model analytical problem: the determination of some endocrine-disrupting phenols in water by liquid chromatography–photometric detection. All the variables involved in the extraction process have been clearly identified and optimized. The new extraction mode allows the determination of these compounds with limits of detection in the range from 0.15 μg/L (for 4-tert-octylphenol) to 2.7 μg/L (for 4-tert-butylphenol) with a relative standard deviation lower than 5.3 % (for 4-tert-butylphenol).     

Near-infrared fluorescence activation probes based on disassembly-induced emission cyanine dye

Currently most of the fluorogenic probes are designed for the detection of enzymes which work by converting the non-fluorescence substrate into the fluorescence product via an enzymatic reaction. On the other hand, the design of fluorogenic probes for non-enzymatic proteins remains a great challenge. Herein, we report a general strategy to create near-IR fluorogenic probes, where a small molecule ligand is conjugated to a novel γ-phenyl-substituted Cy5 fluorophore, for the selective detection of proteins through a non-enzymatic process. Detail mechanistic studies reveal that the probes self-assemble to form fluorescence-quenched J-type aggregate. In the presence of target analyte, bright fluorescence in the near-IR region is emitted through the recognition-induced disassembly of the probe aggregate. This Cy5 fluorophore is a unique self-assembly/disassembly dye as it gives remarkable fluorescence enhancement. Based on the same design, three different fluorogenic probes were constructed and one of them was applied for the no-wash imaging of tumor cells for the detection of hypoxia-induced cancer-specific biomarker, transmembrane-type carbonic anhydrase IX.     

A novel optical thrombin aptasensor based on magnetic nanoparticles and split DNAzyme

In this paper, we report a novel and sensitive optical sensing protocol for thrombin detection based on magnetic nanoparticles (MNPs) and thrombin aptamer, employing split HRP-mimicking DNAzyme halves as its sensing element, which can catalyze the H₂O₂-mediated oxidation of the colorless ABTS into a blue-green product. A single nucleotide containing the recognition element and sensing element is utilized in our protocol. The specific recognition of thrombin and its aptamer leads to the structure deformation of the DNA strands and causes the split of the DNAzyme halves. Therefore, the decrease of absorption spectra can be recorded by the UV–visible Spectrophotometer. DNA-coated MNPs are utilized to separate the interferential materials from the analyst, thus making this assay can be applied in the detection of thrombin in complex samples, such as human plasma. This original, sensitive and cost-effective assay showed favorable recognition for thrombin. The absorbance signals with the concentration of thrombin over a range from 0.5 to 20 nM and the detection limit of thrombin was 0.5 nM. The controlled experiments showed that thrombin signal was not interfered in the presence of other co-existence proteins.     

A Far-Red Molecular Rotor Fluorogenic Trehalose Probe for Live Mycobacteria Detection and Drug-Susceptibility Testing

Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green-emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose-based fluorogenic probe featuring a molecular rotor turn-on fluorophore with bright far-red emission (RMR-Tre). RMR-Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no-wash, low-background fluorescence detection of live mycobacteria. Aided by the red-shifted molecular rotor fluorophore, RMR-Tre exhibited up to a 100-fold enhancement in M. tuberculosis labeling compared to existing fluorogenic trehalose probes. We show that RMR-Tre reports on M. tuberculosis drug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.     

Extremely sensitive sandwich assay of kanamycin using surface-enhanced Raman scattering of 2-mercaptobenzothiazole labeled gold@silver nanoparticles

Herein, we report the development of extremely sensitive sandwich assay of kanamycin using a combination of anti-kanamycin functionalized hybrid magnetic (Fe₃O₄) nanoparticles (MNPs) and 2-mercaptobenzothiazole labeled Au-core@Ag-shell nanoparticles as the recognition and surface-enhanced Raman scattering (SERS) substrate, respectively. The hybrid MNPs were first prepared via surface-mediated RAFT polymerization of N-acryloyl-l-glutamic acid in the presence of 2-(butylsulfanylcarbonylthiolsulfanyl) propionic acid-modified MNPs as a RAFT agent and then biofunctionalized with anti-kanamycin, which are both specific for kanamycin and can be collected via a simple magnet. After separating kanamycin from the sample matrix, they were sandwiched with the SERS substrate. According to our experimental results, the limit of detection (LOD) was determined to be 2 pg mL⁻¹, this value being about 3–7 times more than sensitive than the LOD of previously reported results, which can be explained by the higher SERS activity of silver coated gold nanoparticles. The analysis time took less than 10 min, including washing and optical detection steps. Furthermore, the sandwich assay was evaluated for investigating the kanamycin specificity on neomycin, gentamycin and streptomycin and detecting kanamycin in artificially contaminated milk.     

不同类型探针对免疫层析方法的分析灵敏度影响研究

为研究不同类型探针对免疫层析方法的分析灵敏度影响,该研究以17β-雌二醇(17β-E2)为检测对象,采用磁性纳米材料标记7μg/mL的17β-E2单克隆抗体(检测抗体)制备了传统探针,采用磁性纳米材料分别标记5μg/mL的17β-E2单克隆抗体和5μg/mL的羊抗鼠IgG多克隆抗体(第二抗体)制备了配对探针,采用磁性纳米材料标记14μg/mL的羊抗鼠IgG多克隆抗体制备了间接探针。然后,基于这3种不同类型的探针,分别建立了17β-E2的免疫层析快速检测方法,并对3种17β-E2免疫层析方法的检测灵敏度进行比较。结果显示,基于传统、配对和间接探针的免疫层析方法对17β-E2的检出限分别为1、0.5、0.2 ng/mL,其中基于间接探针的免疫层析方法灵敏度最高,与基于传统探针和配对探针的免疫层析方法相比,检出限分别降低了5倍和2.5倍。该研究刷新了传统免疫层析方法中探针必须是信号材料标记检测抗体的认知,可为其他食品安全危害因子的免疫层析快速检测研究提供有价值的参考。     

Facile synthesis of magnetic one-dimensional polyaniline and its application in magnetic solid phase extraction for fluoroquinolones in honey samples

In this work, we proposed a simple co-mixing method to fabricate magnetic one-dimensional polyaniline (denoted as 1D-PANIs/MNPs). One-dimensional polyanilines (1D-PANIs) and magnetic nanoparticles (MNPs) were prepared by chemical oxidation and solvothermal methods, respectively. When MNPs and 1D-PANIs (with mass ratio 4:1) were co-mixed and vortexed evenly in a solvent (e.g., ethanol, water, acetonitrile), they could assemble into 1D-PANIs/MNPs spontaneously and thus be magnetically separable. To testify the feasibility of 1D-PANIs/MNPs in sample preparation, it was applied as the sorbent for magnetic solid phase extraction (MSPE) of fluoroquinolones (FQs) in honey samples. Under optimized conditions, a rapid, convenient, and efficient method for the determination of four FQs in honey samples by 1D-PANIs/MNPs-based MSPE coupling with high performance liquid chromatography with fluorescence detection (HPLC-FLD) was established. The limits of detection (LODs) for four FQs ranged from 0.4 to 1.4 ng g⁻¹. The intra- and interday relative standard deviations (RSDs) were less than 17.6%. The recoveries of FQs for three spiked honey samples ranged from 86.3 to 121.3%, with RSDs of less than 16.3%.     

Applications of metal nanoparticles/metal-organic frameworks composites in sensing field

Metal nanoparticles (MNPs) possess size-dependent desirable electronic and optical properties while metal-organic frameworks (MOFs) have an edge over extremely large specific surface areas, homogeneous structure, high porosity and remarkable chemical stability. Their combination (MNPs/MOFs) is a novel nanomaterial with broad application prospect in sensing field. To improve performance in sensing applications, we have paid great attention to synergistic effects between the two compositions above. Because of the synergistic effects between MNPs and MOFs, sensors on the basis of MNPs/MOFs composites show significant sensing enhancement with respect to stability, selectivity and sensitivity. In this review, various applications for MNPs/MOFs composites in electrochemical sensing, fluorescent sensing, colorimetric sensing, surface-enhanced Raman scattering sensing and chemiluminescence/electrochemiluminescence sensing are focused and summarized. Besides, the synergistic interactions between MNPs and MOFs was investigated. Finally, based on theoretical information from the reports as well as experimental experience, this review offers the challenges and opportunities for future research on MNPs/MOFs composites.     

Effect of Chemical Charging/Discharging on Plasmonic Behavior of Silver Metal Nanoparticles Prepared using Citrate‐Stabilized Cadmium Selenide Quantum Dots

The thermodynamics and kinetics of the chemical and electrochemical charging of a catalyst surface are very important to understand its applicability as a catalyst material, particularly in redox catalysis. Through the present study, we hereby communicate the results obtained from our detailed investigations related to the effect of chemical charging on the plasmonic behavior of silver metal nanoparticles (Ag MNPs) as redox catalysts. Two different batches of Ag MNPs were prepared through thermally assisted chemical reduction of silver ions. The difference in these batches was the use or not of citrate‐capped cadmium selenide quantum dots (Q‐CdSe) for the reduction of solution‐phase silver ions to their colloidal plasmonic phase. The charge on the surfaces of the Ag MNPs was varied by the chemical electron injection method by using BH₄^? ions from a NaBH₄ solution. The processes of charging and discharging were monitored by using UV/Vis absorption spectroscopy. The impact of the concentration of the reductant on the charging and discharging processes was also investigated. The Ag MNPs were also tested for their voltammetric response, wherein it was observed that it was more difficult to oxidize the Ag MNPs prepared with Q‐CdSe seeds than to oxidize Ag MNPs prepared without Q‐CdSe particles. Our results demonstrate that Q‐CdSe seeds not only enhance the redox catalytic activity of Ag MNPs but also provide stability towards polarization of their plasmonic behavior.     

Determination of Ochratoxin A in apples contaminated with Aspergillus ochraceus by using a microfluidic competitive immunosensor with magnetic nanoparticles

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi of the genus Aspergillus and Penicillium that presents carcinogenic, teratogenic and nephrotoxic properties. In this work, we have developed, characterized and applied an immunoassay methodology comprised of magnetic nanoparticles (MNPs) as platform for immobilizing bioactive materials incorporated into a microfluidic system for rapid and sensitive quantification of Ochratoxin A (OTA) in apples (Red Delicious) contaminated with Aspergillus ochraceus. The sensor has the potential for automation and the detection of OTA was carried out using a competitive indirect immunoassay method based on the use of anti-OTA monoclonal antibodies immobilized on 3-aminopropyl-modified MNPs. The total assay time into the microfluidic competitive immunosensor was 16 min, and the calculated detection limit was 0.05 μg kg(-1). Moreover, the intra- and inter-assay coefficients of variation were below 6.5%. The proposed method can be a very promising analytical tool for the determination of OTA in apparently healthy fruits post-harvest and for its application in the agricultural industry.     

Ultrasensitive detection and molecular imaging with magnetic nanoparticles

Recent advances in nanotechnology have produced a variety of nanoparticles ranging from semiconductor quantum dots (QDs), magnetic nanoparticles (MNPs), metallic nanoparticles, to polymeric nanoparticles. Their unique electronic, magnetic, and optical properties have enabled a broad spectrum of biomedical applications such as ultrasensitive detection, medical imaging, and specific therapeutics. MNPs made from iron oxide, in particular, have attracted extensive interest and have already been used in clinical studies owing to their capability of deep-tissue imaging, non-immunogenesis, and low toxicity. In this Research Highlight article, we attempt to highlight the recent breakthroughs in MNP synthesis based on a non-hydrolytic approach, nanoparticle (NP) surface engineering, their unique structural and magnetic properties, and current applications in ultrasensitive detection and imaging with a special focus on innovative bioassays. We will also discuss our perspectives on future research directions.     

Direct observation of single plasmonic metal nanoparticle reaction in microcolumn with chromatic‐aberration‐free LASER light‐sheet scattering imaging

Direct observation and characterization of individual noble metal nanoparticles (MNPs) and their chemical reactions have attracted much attention owing to their unique physical and chemical properties and extensive applications. To achieve high‐throughput information‐rich evaluation of MNPs, it would be advantageous to combine highly efficient microcolumn separation technology with on‐column high resolution plasmonic imaging technique. Here, with a chromatic aberration‐suppressed supercontinuum laser light‐sheet scattering imaging system and colorimetric detection, we monitored oxidation process of single gold nanorods inside a capillary under gravity driven flow, and observed heterogenous reaction intermediates and pathways for different MNP surface modifications. The results suggest that molecular interactions and bindings with MNPs have a significant impact on their reaction kinetics. This high‐throughput on‐line single particle detection technique could be potentially applied to chemical and biochemical reaction studies of other MNPs.