Streptavidin-modified Magnetic Beads Research Articles

3D DNAzyme walker based electrochemical biosensor for attomolar level microRNA-155 detection

Herein, an ultrasensitive electrochemical biosensor for microRNA-155 (miR-155) detection based on the powerful catalytic and continuous walking signal amplification capability of 3D DNAzyme walker and the gold nanoparticles/graphene aerogels carbon fiber paper-based (AuNPs/GAs/CFP) flexible sensing electrode with excellent electrochemical performance was successfully constructed. In a proof-of-concept experiment, in the presence of miR-155, the DNAzyme strands anchored on the streptavidin-modified magnetic beads (MBs) silenced by locked strands can be activated, thus generating the walking arm of the 3D DNAzyme walker. Meanwhile, the substrate strands modified with Fe-MOF-NH2 nanoparticles were evenly distributed on the surface of MBs and served as tracks of the 3D DNAzyme walker. Once the DNAzyme strand was activated, the catalytic site in the substrate strand can be cleaved in the presence of Mn2+, and a large number of stumps modified with Fe-MOF-NH2 nanoparticles (output@Fe-MOF-NH2) will be generated during the continuous and efficient walking cleavage of the DNAzyme walker, driving the recognition-catalysis-release cycle process for signal amplification. Immediately afterwards, the signal was read out through the base complementary pairing of capture probe (PS) immobilized on the surface of the paper-based flexible sensing electrode AuNPs/GAs/CFP and signal probes output@Fe-MOF-NH2, thus achieving the quantitative detection of miR-155. Under optimal experimental conditions, the designed 3D DNAzyme walker-based biosensor exhibited a relatively lower limit of detection (LOD) of 56.23 aM, with a linear range of 100 aM to 100 nM. Overall, the proposed 3D DNAzyme walker biosensor exhibited good interference and reproducibility, demonstrating a promising future in the field of clinical disease diagnosis.

Magnetically Assisted Immobilization-Free Detection of microRNAs Based on the Signal Amplification of Duplex-Specific Nuclease.

The double specific nuclease (DSN)-based methods for microRNAs (miRNAs) detection usually require the immobilization of DNA probes on a solid surface. However, such strategies have the drawbacks of low hybridization and cleavage efficiency caused by steric hindrance effect and high salt concentration on the solid surface. Herein, we proposed an immobilization-free method for miRNA detection on the basic of DSN-assisted signal amplification. The biotin- and fluorophore-labeled probes were captured by streptavidin-modified magnetic beads through streptavidin-biotin interactions, thus producing a poor fluorescence signal. Once the DNA probes were hybridized with target miRNA in solution to form DNA-miRNA duplexes, DNA stands in the duplexes would be selectively digested by DSN. The released target miRNA could initiate the next hybridization/cleavage recycling in the homogeneous solution, finally resulting in the release of numerous fluorophore-labeled fragments. The released fluorophores remained in solution and emitted strong fluorescence after treatment by the streptavidin-modified magnetic beads. The immobilization-free method achieved the assays of miRNA-21 with a detection limit down to 0.01 pM. It was employed to evaluate the expression levels of miRNA-21 in different cancer cells with satisfactory results.

Highly sensitive homogeneous electrochemiluminescence biosensor for microRNA-155 based on enzyme-free cascade signal amplification and magnetic assisted enrichment

MicroRNA-155 (miR-155) can act as a suitable biomarker for dry eye diseases caused by Sjögren's syndrome. Since the concentration of miR-155 in the clinic samples is low, it is necessary to develop some simple but sensitive detection methods. In this study, a homogeneous electrochemiluminescence (ECL) biosensor for miR-155 detection based on catalyze hairpin assembly (CHA) and hybridization chain reaction (HCR) and magnetic assisted enrichment had been constructed. Firstly, biotin-modified hairpin H1 (bio-H1) had been connected with the streptavidin-modified magnetic beads (SAMBs) to capture target, which can be used to trigger CHA reaction to complete the first signal amplification with the assistance of hairpin H2. Then the stick end of H2 of the CHA production can trigger the HCR reaction of H3 and H4 and generate long double-stranded DNAs (dsDNAs) on the surface of SAMBs to realize the second signal amplification. Ru(phen)32+ can insert into the dsDNAs to produce SAMBs/dsDNA/Ru and act as signal tags, which can be purified by magnetic separation easily, making the background signal significantly reduced. The ECL intensity of the system had a linear relationship with logarithm of miR-155 concentration in the range of 1 fM ∼ 10 nM, with a detection limit as low as 0.63 fM. The proposed biosensor had been applied to monitor miR-155 in clinical samples with satisfied results.

A homogeneous hybridization magnetic biosensor based on electric field assistance for ultrafast nucleic acid detection.

Electrochemical biosensing is a sensitive strategy widely used in the field of nucleic acid detection. However, electrochemical biosensors generally involve time-consuming and labor-intensive probe immobilization processes. In this study, an electrochemical DNA biosensor based on homogeneous hybridization in solution was designed for nucleic acid detection without probe immobilization, which is different from most biosensors. The capture probe, detection probe, and target DNA were hybridized rapidly under an electric field to form a "sandwich" structure within 90 s, and the "sandwich" hybrid could be specifically coupled to streptavidin-modified magnetic beads within 5 min. Finally, the magnetic beads were enriched by using polypyrrole (PPy)/carbon nanotube (CNT)-modified magnetic electrodes and the signal was detected by differential pulse voltammetry (DPV). The magnetic biosensor constructed in this study could detect targets over a good linear dynamic range spanning 100 pM to 100 nM in 400 s, while those involving conventional hybridization methods always take 2 h or more. Because of the specific binding of streptavidin and biotin, this strategy showed high specificity. Taken together, the homogenous hybridization magnetic biosensor constructed with electric field assistance presents a potential diagnostic method for rapid DNA detection and provides a new idea for rapid nucleic acid detection in clinical practice.

Rapid and simple quantitative identification of Listeria monocytogenes in cheese by isothermal sequence exchange amplification based on surface-enhanced Raman spectroscopy.

Foodborne pathogens detection is important to ensure food safety and human health. In this study, we designed a comet structure to rapidly and sensitively detect foodborne Listeria monocytogenes. This method combined isothermal sequence exchange amplification (SEA) and surface-enhanced Raman spectroscopy. Listeria monocytogenes DNA could be rapidly amplified at a constant temperature via SEA with a pair of modified primers, which rendered the precise thermal control instrumentation unnecessary. Efficient SEA amplification generated a large number of DNA duplexes that could be easily captured by streptavidin-modified magnetic bead and AuMB@Ag-isothiocyanate fluorescein antibody (anti-FITC). AuMB@Ag-anti-FITC was used as a signal probe, which generated a significant excitation signal at 1,616 cm-1 for quantitative detection and analysis. The results displayed sensitive detection of L. monocytogenes in cheese from 2.0 × 101 cfu/mL to 2.0 × 106 cfu/mL within 1.0 h with a detection limit of 7.8 cfu/mL. Furthermore, this comet structure displayed the desirable specificity as its specific primers and amplified DNA ends were attached to streptavidin-modified magnetic beads and AuMB@Ag-anti-FITC, respectively. We expected that the method devised would provide a promising new approach to screening for L. monocytogenes and guarantee the microbiological safety of dairy products.

Simultaneous and multiplex detection of exosomal microRNAs based on the asymmetric Au@Au@Ag probes with enhanced Raman signal

Simultaneous and quantitative detection of multiple exosomal microRNAs (miRNAs) was successfully performed by a surface-enhanced Raman scattering (SERS) assay consisting of Raman probes and capture probes. In this design, the asymmetric core-shell structured Au@Au@Ag nanoparticles were first synthesized by layer-by-layer self-assembly method and modified with different Raman molecules and recognition sequences (polyA-DNA) to prepare the surface-enhanced Raman probes. Then, the streptavidin-modified magnetic beads were used to immobilize the biotinylated DNA capture sequences (biotin-DNA) to obtain capture probes. In the presence of target exosomal miRNAs, the Raman probes and capture probes could bind to the target exosomal miRNAs in the partial hybridization manner. Thus, the developed SERS sensor could indicate the target miRNAs levels in the buffer solution. Using breast cancer-related miRNAs as model targets, the limits of detection of this sensor were determined to be 1.076 fmol/L for synthetic miR-21, 0.068 fmol/L for synthetic miR-126, and 4.57 fmol/L for synthetic miR-1246, respectively. Such SERS sensors were further employed to detect the miR-21 in 20% human serum and the extraction solution of exosomes, respectively. Therefore, simultaneous and multiplex detection of cancer-related exosomal miRNAs by this assay could provide new opportunities for further biomedical applications.

A detection method of Escherichia coli O157:H7 based on immunomagnetic separation and aptamers-gold nanoparticle probe quenching Rhodamine B's fluorescence: Escherichia coli O157:H7 detection method based on IMS and Apt-AuNPs probe quenching Rho B' s fluorescence.

This research aimed to detect Escherichia coli O157:H7 in milk based on immunomagnetic probe separation technology and quenching effect of gold nanoparticles to Rhodamine B. Streptavidin-modified magnetic beads (MBs) were combined with biotin-modified antibodies to capture E. coli O157:H7 specifically. Gold nanoparticle (AuNPs) was incubated with sulfhydryl-modified aptamers (SH-Aptamers) to obtain the Aptamers-AuNPs probe. After magnetic beads captured target bacteria and formed a sandwich structure with the gold nanoprobe, Rhodamine B was added into complex to obtain fluorescent signal changes. Our results demonstrated that the established method could detect E. coli O157:H7 in the range of 101-107CFU/mL, and the limit of detection (LOD) was 0.35CFU/mL in TBST buffer (pH = 7.4). In milk simulation samples, the LOD of this method was 1.03CFU/mL. Our research provides a promising approach on the detection of E. coli O157:H7.

A reusable colorimetric assay based on mixed valence state Ce-MOF@Pt nanoparticles for highly sensitive detection of visfatin

In this work, a colorimetric assay for visfatin detection is described. The mixed valence state Ce-MOF (MVCM) modified by platinum nanoparticles (Pt NPs) is used as a novel catalyst. MVCM exhibits excellent intrinsic peroxidase-like catalytic activity due to the spontaneous recycling of the Ce(III)/Ce(IV) system. Pt NPs serve not only as a carrier of the –NH2-modified single strand DNA (S1) but also as a synergistic catalyst of MVCM. The capture probe (S2) attached to the streptavidin-modified magnetic beads (Mag-SA) could combine with the aptamer to form the Mag-SA/S2/aptamer complex. When in the presence of the target visfatin, aptamer specifically combines with the visfatin, which induces the release of Mag-SA/S2 from the Mag-SA/S2/aptamer complex. At this time, the MVCM@Pt/S1 complex connects with the released Mag-SA/S2, which quickly catalyzes the 3,3,3′,3′-tetramethylbenzidine (TMB), leading to a color change. Under optimal conditions, the absorbance increases linearly when the concentration ranges from 1 ng mL−1 to 100 ng mL−1, and the detection limit is as low as 0.11 ng mL−1. Furthermore, Mag-SA/S2 can be reused at least five times by using the uracil-DNA glycosylase (UDG) and an external magnetic field. The proposed method shows satisfying reproducibility, stability, specificity, and sensitivity, and it was successfully applied to detect visfatin in spiked human serum samples. Thus, it has great potential for clinical research, detection, and catalytic applications.

MNAzyme-Catalyzed Amplification Assay with Lanthanide Tags for the Simultaneous Detection of Multiple microRNAs by Inductively Coupled Plasma-Mass Spectrometry.

Quantification of multiple disease-related microRNAs (miRNAs) is of great significance for clinical diagnosis. Based on the simultaneous multiple element detection ability of inductively coupled plasma-mass spectrometry (ICP-MS) and good specificity of multicomponent nucleic acid enzymes (MNAzymes), a novel and simple method based on the MNAzyme amplification strategy and lanthanide labeling coupled with ICP-MS detection was proposed for the sensitive and simultaneous detection of three miRNAs (miRNA-21, miRNA-155, and miRNA-10b). Specifically, a probe consisting of streptavidin-modified magnetic beads (SA-MBs) and three DNA substrates labeled with lanthanide tags (159Tb/165Ho/175Lu) was constructed. In the presence of target miRNAs, three pairs of MNAzymes were assembled where each pair was hybridized with the corresponding miRNA, and then the substrates on the SA-MBs were cleaved by the activated MNAzymes, continuously releasing the fragment with lanthanide tags. The released lanthanide tags in the supernatant were collected after magnetic separation and analyzed by ICP-MS, realizing the simultaneous quantification of multiple miRNAs. The correlation of the lanthanide tag signal with the miRNA concentration fitted well in a linear model in the range of 50-1000 pmol L-1 (miRNA-21) and 50-2000 pmol L-1 (miRNA-155 and miRNA-10b). The limits of detection for three miRNAs were 11-20 pmol L-1, with the relative standard deviations of 2.2-2.7%. The recoveries of target miRNAs in the human serum and HepG-2 cells were in the range of 87.2-111% and 93.3-111%, respectively. Overall, the method is ideal for the simultaneous quantification of multiple miRNAs with advantages of low spectral interference, high sensitivity, good selectivity, and strong resistance to the complex matrix.

An aggregation-induced emission fluorogen/DNA probe carrying an endosome escaping pass for tracking reduced thiol compounds in cells.

The fluorescent nanoprobes for reduced thiol compounds (represented by glutathione, GSH) are constructed based on the aggregation-induced emission (AIE) luminescence mechanism and endosome escape technology. First, a DNA sequence was designed with the decoration of biotin at the 5'-end, disulfide bound in the internal portion, and amino at the 3'-end. The aptamer of the MCF-7 cell was also one of the most important structures in our DNA sequence for the selectivity of MCF-7 cells. We modified streptavidin-modified magnetic beads (MB) with biotin-modified influenza virus hemagglutinin peptide (HA) and biotin-DNA-amino to form MB/DNA/HA. Carboxyl-modified tetraphenylethylene (TPE), an iconic AIE fluorogen, was bonded with amino-modified DNA by covalent interactions (TPE/DNA). Then, the TPE molecule was attached on the outer layer of MB via biotin-modified TPE/DNA to form MB/DNA/HA/TPE. Compared with traditional AIE/biomolecule conjugates, the nanoprobe had an enhanced endosome escape function, due to the assembly of HA. This construction made the intracellular fluorescence response more accurate. In the presence of reduced thiol compounds (take GSH, for example), the disulfide bond on the DNA was reduced by thiol-disulfide exchange reactions and the TPE molecule was released into the solution. The shedding TPE molecule was more hydrophobic than TPE/DNA and the conversion of TPE/DNA to shedding TPE could lead to the aggregation of the TPE fluorogen. Thus, its fluorescence was enhanced. Under the optimized condition, the fluorescence intensity increased with the increase in concentration of GSH' ranging from 1.0 × 10-9M to 1.0 × 10-5M' and the detection limit was 1.0 × 10-9M. The relative standard deviation (RSD) was calculated to be 3.6%. The recovery in cell homogenate was from 94.5 to 102.7%. The nanoprobe provided a way for the detection of reduced thiol compounds in MCF-7 cells. We envision that, in the near future, our strategy of DNA-instructed AIE could be widely applied for biosensing and bioimaging in vitro and even in vivo with dramatically enhanced sensitivity. Graphical Abstract.

A Novel Cell-Assisted Enhanced Chemiluminescence Strategy for Rapid and Label-Free Detection of Tumor Cells in Whole Blood.

In this study, an interesting phenomenon was found where cells (including tumor and normal cells) managed to significantly enhance chemiluminescence (CL) signals. The possible reaction mechanism may be that cells can be severely damaged by CL substrates, and the released contents, possibly proteins (such as cytochrome c), can remarkably magnify CL owing to the increased production of singlet oxygen. More importantly, based on the above phenomena, a novel cell-assisted enhanced CL strategy was proposed for the rapid and label-free detection of tumor cells. The complexes of aptamer sgc8c and streptavidin-modified magnetic beads were employed to recognize and isolate target tumor cells from whole blood. The enhanced CL intensity, which was triggered directly by the captured cells, was measured. The proposed strategy exhibited a good detection performance with a linear range from 200 to 10,000 cells/mL. The analysis can be finished in ∼30 min, and the limit of detection was down to 100 cells/mL. The recoveries and relative standard deviations were 97.81-102.71% and 3.46-12.71%, respectively. Moreover, the established method can successfully distinguish the leukemia patients from healthy people. Therefore, it provides a novel, rapid, and simple method for the determination of tumor cells, which can be used in further practice.

Ultra-sensitive and chemiluminescent detection of telomerase activity via handheld luminometer

The point-of-care testing (POCT) of disease biomarker is crucial and in great demand. Herein, the activity of tumor biomarker, telomerase, was simply and sensitively detected via handheld luminometer based on the horseradish peroxidase (HRP) catalyzed luminol/H2O2 chemiluminescence (CL) reaction. Biotinylated telomerase substrate (TS) primer was loaded onto the streptavidin-modified magnetic beads (MBs) surface via biotin-avidin interaction. In the presence of telomerase, TTAGGG repeated units were progressively added to the 3′ end of TS to form a long telomeric DNA elongation product (TEP) which will hybridize with multiple short HRP-labeled complementary DNA (HRP-cDNA), leading to the enrichment of HRP on the surface of MBs. An obvious chemiluminescence was observed when MBs complexes were mixed with luminol/H2O2/p-Iodophenol (PIP) mixture. The CL intensity was linearly related with the logarithm of the number of HeLa cells in the range of 10–1000 cells. By combining the self-amplification of telomerase with the high sensitivity of CL, the ultra-sensitive detection of telomerase activity was achieved, and the detection limit was estimated down to 9 cells. Detection of telomerase activity in serum achieved satisfactory results. The replacement of the commercial fluorescein-luciferase test swab by luminol/H2O2/PIP solution led to a more stable and cheaper POC assay, which holds great potential for exploring facile, sensitive and reliable POCT.

Detection of DNA 3'-phosphatase activity based on exonuclease III-assisted cascade recycling amplification reaction

DNA 3'-phosphatase is an essential enzyme, which plays a pivotal role in repairing DNA damage. The peculiar activity of DNA 3'-phosphatase has been proved to associate with a variety of human pathologies. Therefore, sensitive determination of DNA 3'-phosphatase is necessary for clinical diagnosis and therapy. Here, we develop a simple, sensitive, and specific fluorescent biosensor including three DNA chains of hairpin DNA1, hairpin DNA2 and fluorescence probe DNA (FP) for detecting the activity of DNA 3'-phosphatase. First, biotin-modified hairpin DNA1 binds with streptavidin-modified magnetic beads (MB) to get MB-DNA1. DNA 3'-phosphatase can hydrolyze phosphate groups on MB-DNA1 to form hydroxyl groups, which leads to the polymerization extension and nicking endonuclease cleavage reaction to obtain the trigger DNA1 fragment (tDNA1). Next, two cyclic amplification reactions are designed. In cycle I, the tDNA1 hybridizes with the hairpin DNA2, which leads the hairpin structure of DNA2 opened and the fluorescence signal of 6-carboxy-fluorescein (FAM) labeled on hairpin DNA2 turned on. This cyclic reaction is amplified by exonuclease III (Exo III). At the same time, the trigger DNA2 fragment (tDNA2) is obtained. In cycle II, similarly, the tDNA2 hybridizes with FP. Thus, the fluorescence signal of FAM labeled on FP released, which multiplies with the fluorescence signal from cycle I. Finally, this strategy is applied to determine two typical DNA 3'-phosphatases including T4 polynucleotide kinase (T4 PNK) and alkaline phosphatase (ALP) with the detection limit (LOD) of 0.0033 and 0.00037 U/mL, respectively. The method provides a promising platform to evaluate the DNA 3'-phosphatase activity in the complicated biological samples and can be potentially applied in the relevant fields such as biomedical research, drug discovery and clinical diagnosis.

A highly sensitive assay of DNA based on inductively coupled plasma mass spectrometry detection with gold nanoparticle amplification and isothermal circular strand-displacement polymerization reaction

In this work, a simple, sensitive and specific assay for DNA was proposed by combining inductively coupled plasma mass spectrometry (ICP-MS) detection with gold nanoparticle (AuNPs) amplification and isothermal circular strand-displacement polymerization reaction (ICSDPR). First, AuNPs were decorated with hairpin-structured DNA (HP-DNA) through Au–S bond to form the AuNPs probe. The ICSDPR was conducted on AuNPs probe in a homogeneous phase to realize the dual amplification and simplify the analytical process at the same time. By using a biotin modified primer, AuNPs were connected with biotins after the ICSDPR, then captured by the streptavidin modified magnetic beads (SA-MBs), and finally detected by ICP-MS. Many key factors including probe volume, hybridization time, polymerase amount, primer concentration, enzyme reaction time, SA-MBs capture time and desorption time were optimized. Under the optimized condition, the proposed method could detect target DNA as low as 45 zmol (8.9 fM in 5 μL) in a relative short time (about 4.5 h) with good specificity, and the linear range of this method is 0.1–10000 pM, the relative standard deviations are in the range of 3.6–6.4%. The proposed method was applied for determination of target DNA in human serum samples, the recovery for the spiked human serum samples is in the range of 84–120%. It demonstrates a good application potential of the developed method for biological studies and clinical diagnosis of human pathogenic diseases.

Electrochemical determination of the activity of DNA methyltransferase based on the methyl binding domain protein and a customized modular detector.

An electrochemical method is described for the determination of the activity of the DNA methyltransferase (MTase). The assay was based on the use of a commercially available customized electromagnetic modular detector, which consisted of a magnetic switch, electrical connectors and a screen-printed electrode modified with graphene oxide. The biotinylated single-strand DNA (ss-DNA) S1 was absorbed by streptavidin-modified magnetic beads (MBs) via streptavidin-biotin interaction. The biotinylated ss-DNA S1 was hybridized with the complementary ss-DNA S2. After the symmetrical sequences 5'-CCGG-3' of the duplex DNA (ds-DNA) were methylated by M. SssI CpG methyltransferase (M. SssI MTase), the symmetrical sequences 5'-CCGG-3' in the ds-DNA were recognized by glutathione S-transferase (GST) tagged methyl CpG binding protein 2 (MeCP2). The unmethylated 5'-CCGG-3' sequences were specifically cleaved by HpaII restriction endonuclease. After magnetic separation and washing, HRP-labeled GST tag monoclonal antibody and H2O2 were used as a tracer label and enzyme substrate, respectively. Electrochemical measurement was carried out atpH7.4in the presence of 50μM thionine and 0.5mM H2O2. Stepwise changes in the microscopic features of the SPE surface upon the formation of each layer were studied by scanning electron microscopy. Cyclic voltammetry and differential pulse voltammetry were used to characterize the electrochemical behavior of the different modified electrodes. Under the optimal conditions, the activity of M. SssI MTase can be determined in the activity range of 0.5-125 unit·mL-1 with a detection limit of 0.2 unit·mL-1 (at an S/N ratio of 3). The sensitivity of the immunoassay is 0.489μA·μM-1·cm-2. Graphical abstract Schematic presentation of the electrochemical immunosensor for the determination of the activity of M. SssI CpG methyltransferase (M. SssI MTase). It is based on an electromagnetic modular detector and the use ofglutathione S-transferase tagged methyl CpG binding protein 2 (GST-MeCP2).

Electrochemical sandwich immunoassay for human epididymis-specific protein 4 using a screen-printed electrode modified with graphene sheets and gold nanoparticles, and applying a modular magnetic detector device produced by 3D laser sintering

The authors describe an electrochemical sandwich immunoassay for the human epididymis-specific protein 4 (HE4). A commercially available electromagnetic detector device and a screen-printed electrode (SPE) modified with graphene sheets and gold nanoparticles were used to fabricate the detector. First, a nanocomposite suspension consisting of graphene sheets and gold nanoparticles was deposited onto the SPE. This results in an enlarged electrochemically active area and improves electron transfer. Next, biotinylated monoclonal antibody against HE4 (anti-HE4) was bound to streptavidin-modified magnetic beads via biotin-avidin binding. Nonspecific binding sites were blocked with bovine serum albumin. Stepwise changes in the microscopic features of the surfaces and electrochemical properties upon the formation of each layer were studied by scanning electron microscopy, transmission electron microscopy and cyclic voltammetry. After magnetic separation and washing, the biotinylated anti-HE4 beads were deposited on the SPE, which then was inserted into the electromagnetic modular detector. Following the immunoreaction with HE4, a sandwich-type immunoassay was performed on the SPE using horseradish peroxidase (HRP)-labeled HE4 as a tracer label. Electrochemical detection was carried out after addition of H2O2 as a substrate for HRP. Under optimal conditions, differential pulse voltammetry can detect HE4 in the 0 to 400 pM concentration range with a detection limit of 0.5 pM (at an S/N ratio of 3). The system was applied to the determination of HE4 with good accuracy and selectivity.

Multifunctional assembly of micrometer-sized colloids for cell sorting.

Compared to the extensively studied nanometer-sized colloids, less attention has been paid to the assembly of micrometer-sized colloids with multifunctional characteristics. To address this need, a bottom-up approach is developed for constructing self-assemblies of micrometer-sized magnetic colloids possessing multifunctionality, including magnetic, optical, and biological activities. Biotinylated oligo (p-phenylene vinylene) (OPV) derivatives are designed to mediate the self-assembly of streptavidin-modified magnetic beads. The optical element OPV derivatives provide a fluorescence imaging ability for tracing the assembly process. Target cells can be recognized and assembled by the colloidal assembly with bioactive element antibodies. The colloidal assembly reveals better cell isolation performance by its amplified magnetic response in comparison to monodisperse colloids. The self-assembly of micrometer-sized magnetic colloids through a combination of different functional ingredients to realize multifunction is conceptually simple and easy to achieve.

Ultrasensitive detection of DNA and RNA based on enzyme-free click chemical ligation chain reaction on dispersed gold nanoparticles.

An ultrasensitive colorimetric DNA and RNA assay using a combination of enzyme-free click chemical ligation chain reaction (CCLCR) on dispersed gold nanoparticles (GNPs) and a magnetic separation process has been developed. The click chemical ligation between an azide-containing probe DNA-modified GNP and a dibenzocyclooctyne-containing probe biotinyl DNA occurred through hybridization with target DNA (RNA) to form the biotinyl-ligated GNPs (ligated products). Eventually, both the biotinyl-ligated GNPs and target DNA (RNA) were amplified exponentially using thermal cycling. After separation of the biotinyl-ligated GNPs using streptavidin-modified magnetic beads, the change in intensity of the surface plasmon band at 525 nm in the supernatants was observed by UV/vis measurement and was also evident visually. The CCLCR assay provides ultrasensitive detection (50 zM: several copies) of target DNA that is comparable to PCR-based approaches. Note that target RNA could also be detected with similar sensitivity without the need for reverse transcription to the corresponding cDNA. The amplification efficiency of the CCLCR assay was as high as 82% due to the pseudohomogeneous reaction behavior of CCLCR on dispersed GNPs. In addition, the CCLCR assay was able to discriminate differences in single-base mismatches and to specifically detect target DNA and target RNA from the cell lysate.

Turn-on chemiluminescent sensing platform for label-free protease detection using streptavidin-modified magnetic beads

We report a label-free streptavidin-modified magnetic beads (SA-MBs)-based sensing platform for turn-on chemiluminescent (CL) detection of protease using trypsin as model analyte. In the assay, a biotinylated peptide containing an arginine and a terminal cysteine was used as the substrate of trypsin. Upon adding the peptide into a basic luminol-NaIO4 solution, the terminal cysteine induced a strong CL signal. Surprisingly a much lower CL was emitted when the peptide was immobilized on the surface of SA-MBs. Based on this phenomenon, we designed a turn-on CL sensing system for protease using trypsin as model and its inhibitors screening. In the absence of trypsin, the peptide was coupled to the SA-MBs surface, resulting in a low CL background. Upon the addition of trypsin, the peptide can be catalytically hydrolyzed at the C-terminus of arginine, resulting in the formation of free cysteine-containing residues and subsequent CL recovery with the addition of luminol and NaIO4. The simple method does not require washing or separating procedures. Trypsin at a concentration as low as 10pM can be assayed using this new CL sensing system. Additionally, the proposed method can be employed for screening the inhibitors of trypsin. This new sensing strategy could be easily extended to assay other proteases by simply changing the peptide substrate.

Microbead-Based Ligase Detection Reaction Assay Using a Molecular Beacon Probe for the Detection of Low-Abundance Point Mutations

A microbead-based ligase detection reaction (LDR) assay using a molecular beacon probe was developed for the facile and rapid detection of point mutations present in low copy numbers in a mixed population of wild-type DNA. Biotin-tagged ligation products generated in the LDR were captured on the surface of streptavidin-modified magnetic beads for purification and concentration. The resulting product-tethered microbeads were combined with a molecular beacon probe solution, and the suspension was directly flowed into a capillary. The microbeads were accumulated in a confined space within the capillary using a bar magnet. The packed bead sample was then scanned by a fluorescence scanning imager to detect the presence of any mutations. With the developed methodology, we were able to successfully detect one cancer mutation in a mixture of 400 wild-type templates (t test at 95% confidence level). Furthermore, the post-LDR processing, typically the most laborious and time-consuming step in LDR-based mutation detection assays, could be carried out much more rapidly (approximately 20 min). This was enabled by the simple bead and fluid manipulations involved in the present assay.