Aptamer-modified Magnetic Beads Research Articles

Manipulation of magnetic beads for actively capturing Vibrio parahaemolyticus and nucleic acid based on microfluidic system.

Rapid biological detection of pathogen micro-organisms has attracted much attention for practical biomedical applications. Despite the development in this field, it is still challenging to achieve simple and rapid biological detection using the microfluidic method. Herein, we propose a novel strategy of biological detection that combines precise detection control of the capillary microfluidic chip and versatile manipulation of magnetic beads. The microfluidic chip was fabricated via laser cutting, which utilized capillary pressure to realize rapid passive injection of liquid samples. Under an external magnetic field, the aptamer-modified magnetic beads were actuated to mix with Vibrio parahaemolyticus (V. parahaemolyticus) and its nucleic acid in the capillary microfluidic chip for rapid selective capture and detection, which could be achieved within 40 min. The experimental results demonstrated that V. parahaemolyticus could be captured using on-chip immunomagnetic beads with a high efficiency and significantly enhanced detection value. Due to these superior performances, the capillary microfluidic system, based on the manipulation of magnetic beads, demonstrated great potential for automatic biological detection.

A Cascaded Phase-Transfer Microfluidic Chip with Magnetic Probe for High-Activity Sorting, Purification, Release, and Detection of Circulating Tumor Cells.

Microfluidic chips have emerged as a promising tool for sorting and enriching circulating tumor cells (CTCs) in blood, while the efficacy and purity of CTC sorting greatly depend on chip design. Herein, a novel cascaded phase-transfer microfluidic chip was developed for high-efficiency sorting, purification, release, and detection of MCF-7 cells (as a model CTC) in blood samples. MCF-7 cells were specifically captured by EpCAM aptamer-modified magnetic beads and then introduced into the designed cascaded phase-transfer microfluidic chip that consisted of three functional regions (sorting, purification, and release zone). In the sorting zone, the MCF-7 cells moved toward the inner wall of the channel and entered the purification zone for primary separation from white blood cells; in the purification zone, the MCF-7 cells were transferred to the phosphate-buffered saline flow under the interaction of Dean forces and central magnetic force, achieving high purification of MCF-7 cells from blood samples; in the release zone, MCF-7 cells were further transferred into the nuclease solution and fixed in groove by the strong magnetic force and hydrodynamic force, and the continuously flowing nuclease solution cleaved the aptamer on the trapped MCF-7 cells, causing gentle release of MCF-7 cells for subsequent inductively coupled plasma mass spectrometry (ICP-MS) detection or further cultivation. By measurement of the endogenous element Zn in the cells using ICP-MS for cell counting, an average cell recovery of 84% for MCF-7 cells was obtained in spiked blood samples. The developed method was applied in the analysis of real blood samples from healthy people and breast cancer patients, and CTCs were successfully detected in all tested patient samples (16/16). Additionally, the removal of the magnetic probes on the cell surface significantly improved cell viability up to 99.3%. Therefore, the developed cascaded phase-transfer microfluidic chip ICP-MS system possessed high integration for CTCs analysis with high cell viability, cell recovery, and purity, showing great advantages in early clinical cancer diagnosis.

Hybridization chain reaction-enhanced electrochemically mediated ATRP coupling high-efficient magnetic separation for electrochemical aptasensing of cardiac troponin I

The sensitive and accurate detection of cardiac troponin I (cTnI) as a gold biomarker for cardiovascular diseases at an early stage is crucial but has long been a challenge. In this study, we presented such an electrochemical (EC) aptasensor by combining hybridization chain reaction (HCR)-enhanced electrochemically mediated atom transfer radical polymerization (eATRP) amplification with high-efficient separation of magnetic beads (MBs). Aptamer-modified MBs empowered effective recognition and separation of cTnI from complex samples with high specificity. The specific binding of cTnI and aptamer could release triggered DNA (T-DNA) into solution to drive an HCR process, which produced plentiful active sites for eATRP initiators labeling followed by initiating eATRP process. With the development of eATRP, a great many of electroactive polymer probes were continually in situ formed to generate amplified current output for signal enhancement. Compared to no amplification, HCR-enhanced eATRP promoted the signals by ∼10-fold, greatly improving detection sensitivity for low-abundant cTnI analysis. Integrating MBs as capture carriers with HCR-enhanced eATRP as amplification strategy, this EC aptasensor achieved a low detection limit of 10.9 fg/mL for cTnI detection. Furthermore, the reliable detectability and anti-interference were confirmed in serum samples, indicating its promising application toward early diagnosis of cardiovascular diseases.

Construction of Liquid Crystal-Based Sensors Using Enzyme-Linked Dual-Functional Nucleic Acid on Magnetic Beads.

The development of liquid crystal (LC)-based sensors with superior performances such as high portability, excellent stability, great convenience, and remarkable sensitivity is highly demanded. This work proposes a new strategy for constructing the LC-based sensor using enzyme-linked dual-functional nucleic acid (d-FNA) on magnetic beads (MBs). The detection of kanamycin (KA) is demonstrated as a model. Acetylcholinesterase (AChE) is assembled onto the KA aptamer-modified MBs with a d-FNA strand that consists of an AChE aptamer and the complementary sequence of a KA aptamer. As the specific recognition of KA by its aptamer triggers the release of AChE from the MBs, the myristoylcholine (Myr) solution after incubation with the MBs causes the black image of the LCs due to the formation of the Myr monolayer at the aqueous/LC interface. Otherwise, in the absence of KA, AChE is still decorated on the MBs and causes the hydrolysis of Myr. Therefore, a bright image of LCs is obtained. The detection of KA is successfully achieved with a lower detection limit of 48.1 pg/mL. In addition, a thin polydimethylsiloxane (PDMS) layer-coated glass and a portable optical device are used to improve the stability and portability of the LC-based sensor to advance potential commercial applications. Furthermore, the detection of KA in milk with a portable device is demonstrated, showing the potential of the proposed enzyme-linked LC-based sensor.

Hybridized Chain Reaction-Amplified Alkaline Phosphatase-Induced Ag-Shell Nanostructure for the Sensitive and Rapid Surface-Enhanced Raman Scattering Immunoassay of Exosomes.

Exosomes are small extracellular vesicles that can be utilized as noninvasive biomarkers for diagnosis and treatment of cancer and other diseases. This study reports on a strategy involving a hybridized chain reaction-amplified chain coupled with an alkaline phosphatase-induced Ag-shell nanostructure for the ultrasensitive and rapid surface-enhanced Raman scattering immunoassay of exosomes. Exosomes from prostate cancer were captured using prostate-specific membrane antigen aptamer-modified magnetic beads; then, the hybridized chain reaction-amplified chain was released, incorporating a large number of functional moieties with signal amplification effects. Moreover, the steps of traditional immunoassay were simplified using magnetic materials, and the rapid, sensitive, and accurate detection of exosomes was achieved. Results could be obtained within 40 min with a detection limit of 19 particles/μL. Furthermore, the sera of human prostate cancer patients could be easily distinguished from those of healthy controls, highlighting the potential use of exosome analysis in clinical diagnostics.

In silico selection of an aptamer for the design of aptamer-modified magnetic beads bearing ferrocene co-immobilized label for capacitive detection of acetamiprid.

In silico evaluation of aptamer/target interactions can facilitate the development of efficient biosensor with high specificity and affinity. In this work, we present in silico, i.e. structural similarity, molecular docking and molecular dynamics selection of the aptamer with sufficient binding properties for acetamiprid (ACE), a nicotine-like pesticide, and its use to design aptamer-modified magnetic beads bearing ferrocene co-immobilized label for capacitive detection of ACE. Taking advantages of the aptamer higher stability and binding affinity, the specific properties of magnetic beads and the redox properties of ferrocene moiety, the developed aptasensor showed promising analytical performances for ACE detection, using electrochemical capacitance spectroscopy, with a linear response ranging from 1fM to 100 pM and a limit of detection of 0.94fM(S/N=3). Furthermore, it was successfully applied to detect ACE in fortified tomatoes samples, proving a promising approach for routine detection of pesticide in real agricultural samples.

An excellent colorimetric aptasensor integrating multifunctional SNAs and TdT-induced dual signal amplification for rapid sensitive detection of exosomes

Exosome-based liquid biopsy has shown great potential in the diagnosis and prognosis of tumors, thus developing a convenient and reliable detection method is crucial for its clinical application. Herein, we construct a separable visual aptasensor for the sensitive detection of exosomes by using double spherical nucleic acids (DSNAs) and terminal deoxynucleotidyl transferase (TdT). In this system, CD63 aptamer-modified magnetic beads (MNPs-AptCD63, the first spherical nucleic acids) could efficiently capture exosomes, and avoid the aggregation of gold nanoparticles (GNPs) induced by complex components in the reaction system through magnetic separation. As the second spherical nucleic acids, abundant nucleolin aptamer-modified GNPs (GNPs-Aptnucleolin) are used to specially identify exosomes. Then, those nucleolin aptamers can further produce long polyT sequences by the extension of TdT and induce polyA release from the GNPs-polyA (polyA-attached gold nanoparticles) by hybridization, thus resulting in the salt-induced aggregation of GNPs to produce color change for visual analysis of exosomes. Based on the above ingenious designed strategies, this method achieved a limit of detection (LOD) as low as 45 particles/μL, and showed accurate visual analysis for the clinical samples. We expect this method to provide a new opportunity for the exosomes-based point-of-care diagnosis of tumors.

Rapid detection of Saccharomyces cerevisiae with boronic acid-decorated multivariate metal-organic frameworks and aptamers.

Liquor brewing is a classic solid-substrate fermentation process with a unique brewing microbiome. As one of the most common fungi, Saccharomyces cerevisiae ferments saccharides and has been extensively applied in brewing production. Here, we present the facile fabrication of a selective, sensitive, and integrated fluorescent biosensor for S. cerevisiae detection. The proposed biosensor used aptamer-modified magnetic beads to specifically capture S. cerevisiae, and the enriched fungi were recognized and detected with boronic acid-decorated multivariate metal-organic frameworks. The biosensor allows rapid quantification of S. cerevisiae in the range of 10-106 CFU mL-1, showing excellent specificity and repeatability, and maintaining stable biosensing performance in long-term storage. The analytical ability of the proposed biosensor was successfully verified in distilled yeast and fermented grain samples spiked with S. cerevisiae.

Highly sensitive photoelectrochemical detection of cancer biomarkers based on CdS/Ni-CAT-1 nanorod arrays Z-scheme heterojunction with spherical nucleic acids-templated copper nanoclusters as signal amplification

Ultrasensitive and accurate detection of low-abundance cancer markers is critical for early diagnosis of cancer. To date, developing high-performance photoelectrode and efficient signal amplification strategies remain core challenges for photoelectrochemical bioanalysis. Herein, a CdS/conductive metal-organic frameworks nickel-catecholates nanorod (Ni-CAT-1 NR) arrays Z-scheme heterojunction was developed as a high-performance photoelectrode for split-type photoelectrochemical aptasensing of carcinoembryonic antigen (CEA, as a model cancer marker) with advanced spherical nucleic acids-templated copper nanoclusters (SNAs-Cu NCs) as signal amplification. The Ni-CAT-1 NR arrays not only match with the energy band of CdS, but also act as a direct pathway for electron transfer to the electrode surface, reducing the photogenerated charges recombination rate. In the presence of CEA, aptamer-modified magnetic beads and aptamer-anchored SNAs-Cu NCs reacted with the target to form a sandwich-like structure and plenty of Cu2+ released from SNAs-Cu NCs by acidolysis could react with CdS/Ni-CAT-1 NR arrays to generate trapping site, reducing the photocurrent. Under optimal conditions, the prepared aptasensor showed a wide linear range from 0.01 pg mL−1-1.0 ng mL−1, a low detection limit of 0.003 pg mL−1, and excellent stability and accuracy, demonstrating a promising platform for biomarkers detection.

Magnetic bead-based electrochemical and colorimetric assays of circulating tumor cells with boronic acid derivatives as the recognition elements and signal probes

Simple, sensitive and rapid detection of circulating tumor cells (CTCs) is of great importance for early diagnosis and therapy of cancers. Overexpression of sugar units on cell surface is related to the phenotypes of many cancers. Based on the boronate ester interaction, we reported the electrochemical and colorimetric detection of CTCs with high simplicity and sensitivity. Specifically, ferroceneboronic acid (FcBA) can be measured by differential pulse voltammetry and 4-mercaptophenylboronic acid (MPBA) can induce the aggregation and color change of gold nanoparticles (AuNPs). CTCs captured by the aptamer-modified magnetic beads (Apt-MBs) can sequestrate FcBA or MPBA molecules by the formation of boronate ester bonds, thus leading to the decrease in the electrochemical signal of FcBA or preventing the MPBA-triggered aggregation of AuNPs. Due to the overexpression of sugar groups on the surface of CTCs, the amplification-free methods exhibited high sensitivity and obviated the use of additional antibody or aptamer for the recognition of captured cells. With MCF-7 cancer cell as the model, 50 cells can be readily determined by the electrochemical and colorimetric methods. The proposed strategy is valuable for probing of cell glycosylation and designing of novel sensing devices for detection of sugar-containing biological macromolecules and cells.

Development of a highly sensitive detection method for TTX based on a magnetic bead-aptamer competition system under triple cycle amplification

We have established an assay that relies on aptamer and isothermal amplification for the tetrodotoxin (TTX)detection. The method uses triple cycle amplification (strand displacement amplification combined with catalytic hairpin assembly) and fluorescent reporter as an output signal. Free TTX and cDNA compete for binding to aptamer-modified magnetic beads. The cDNA collected by magnetic separation then used as a primer to trigger triple cycle amplification to obtain more ssDNA. The ssDNA combined with the reporter probe, and the original quenched fluorescence can be recovered. In addition, a linear relationship between fluorescence spectrum and different target concentrations is revealed. This method allows TTX to be detected by fluorometry with a detection limit as low as 0.265 pg mL−1. It was applied to clams and shellfish, achieving recoveries ranging from 100% to 107.33% and 99.67%–116.67%, respectively. The results were consistent with the commercial TTX ELISA kit. This assay is highly sensitive, reliable and has a good specificity. Therefore, it provides a better alternative to the standard method for quantitative detection of TTX.

Ultrasensitive competitive detection of patulin toxin by using strand displacement amplification and DNA G-quadruplex with aggregation-induced emission

A novel sensitive assay was established by using strand displacement amplification (SDA) and DNA G-quadruplex with aggregation-induced emission (AIE) for the detection of patulin (PAT) toxin. The complementary DNA (cDNA) of the aptamer and PAT competed for binding to aptamer-modified magnetic beads. The cDNA was obtained by magnetic separation and used as a primer in SDA to produce a large amount of G-base single-stranded DNA (ssDNA). They can form the G-quadruplex to be combined with the AIE of TTAPE dye, which features a special combination of G-quadruplex that amplify the fluorescent signals. This work can reach a lower detection limit of 0.042 pg mL−1 with a wide linear range of 0.001–100 ng mL−1 for PAT detection than other methods. The results also showed good recoveries of 97.8%–104% and 101.7%–105.3% in spiked apple and grape juices, respectively. The assay used for the detection of PAT exhibits high sensitivity and good specificity. It also provides a stable and reliable platform for detecting other small-molecule toxins.

Microchip electrophoretic detection of bacterial lipopolysaccharide based on aptamer-modified magnetic beads and polymerase chain amplification

Bacterial lipopolysaccharides (LPS) are endotoxins, which can cause fever, inflammation, cell and tissue damage, irreversible septic shock and death in mammals as well as other organisms. Herein, a highly sensitive and selective strategy was proposed for detecting LPS using microchip electrophoresis (MCE) based on aptamer-modified magnetic beads and polymerase chain amplification. In this strategy, the biotinylated aptamer for the LPS (LBA) was hybridized partially with the complementary DNA (cDNA), then the double-stranded DNA obtained was immobilized onto the magnetic beads. In the presence of target LPS, the target-aptamer complex was formed and the cDNA would be freed. After a magnetic separation, the cDNA was released into the supernatant and subsequently triggered the polymerase chain amplification. This process eventually generated a large number of output DNA, which could be quantitatively detected by MCE. With the polymerase chain amplification, this designed protocol provided an ultrasensitive detection of LPS down to the 1.1 × 10−14 g/mL with a linear range of 5 orders of magnitude. The proposed method also exhibited a high specificity toward LPS in the presence of other common interfering substances. Moreover, the assay showed a good practical application for LPS determination in serum samples.

Femtomolar electroanalysis of a breast cancer biomarker HER-2/neu protein in human serum by the cellulase-linked sandwich assay on magnetic beads

In cancer diagnostics, specific analysis of blood-circulating proteins biomarkers of cancer is often complicated both by their inherently low concentrations and by strong interference from serum/blood proteins. Here, we report a simple and robust electrochemical cellulase-linked sandwich assay on magnetic beads (MBs) for fM-sensitive analysis of the Human Epidermal growth factor Receptor-2 HER-2/neu protein that is over-expressed in most aggressive breast cancers. In the assay, a sandwich is assembled by capturing HER-2/neu on either antibody (Ab) or aptamer-modified MBs accompanied by reaction with the second Ab or aptamer labelled with cellulase. On application of the sandwiches assembled on MBs onto a cost-effective graphite electrode modified with an insulating nitrocellulose film, the cellulase label digests the film. This results in the pronounced changes in the electrical properties of the modified electrodes. The chronocoulometrically-measured extent of the produced changes was proportional to the 10−15-10−10 M HER-2/neu in the analyzed samples, and down to 1 fM of HER-2/neu could be detected in human serum samples in an overall less than 3 h assay. The developed simple and electrochemically label-free methodology is general and can be easily adapted for testing of any other protein.

Competitive fluorometric assay for the food toxin T-2 by using DNA-modified silver nanoclusters, aptamer-modified magnetic beads, and exponential isothermal amplification.

The authors describe an aptamer based assay for the mycotoxin T-2. The method is making use of exponential isothermal amplification reaction (EXPAR) and fluorescent silver nanoclusters (AgNCs). Free T-2 and cDNA (which is a DNA that is partially complementary to the aptamer) compete for binding to aptamer-modified magnetic beads. The cDNA collected by magnetic separation can be used as a primer to trigger EXPAR to obtain ssDNA. The C-base-rich ssDNA binds and reduces Ag(I) ion to form fluorescent AgNCs. Fluorescence is measured at excitation/emission wavelengths of 480/525nm. T-2 can be detected by fluorometry with a detection limit as low as 30fg·mL-1. The method was applied to analyse spiked oat and corn, and the average recoveries ranged from 97.3 to 102.3% and from 95.9 to 107.5%, respectively. The results were in good agreement with data of the commercial ELISA kit. The assay is highly sensitive, has a wide analytical range, good specificity and reliable operation. It provides a promising alternative for the standard method for quantitative detection of T-2. Graphical abstract Schematic presentation of fluorometric assay for T-2 based on aptamer-functionalized magnetic beads exponential, isothermal amplification reaction (EXPAR) and fluorescent silver nanoclusters (AgNCs).

An aptamer-based new method for competitive fluorescence detection of exosomes.

Exosomes have been recognized as promising sources of biomarkers for early cancer diagnosis due to their important role in the occurrence and metastasis of cancer, and so the development of a sensitive low-cost detection method for exosomes is highly desirable. In this paper, we report a fluorescence method for the competitive detection of exosomes based on an aptamer specific to CD63 (an exosome transmembrane protein). Aptamer-modified magnetic beads were hybridized with a Cy3-labeled short sequence complementary to a region of the aptamer. In the presence of exosomes, the CD63 on the exosomes bound to the aptamer, resulting in the shedding of the short sequence into the supernatant. The quantity of the exosomes could be estimated by detecting the fluorescence intensity in the supernatant. This method could detect exosomes at a concentration as low as 1.0 × 105 particles per μL under optimal conditions, and the feasibility of the method for exosome detection in complex clinical samples was also proved using simulated serum samples. The detection cost and difficulty are significantly reduced compared to conventional methods, while ensuring sensitivity, and so this method provides a basis for subsequent exosome detection in specific cancer cells.

A colorimetric aptasensor for the antibiotics oxytetracycline and kanamycin based on the use of magnetic beads and gold nanoparticles.

An aptamer based assay is presented for the determination of the antibiotics oxytetracycline (OTC) and kanamycin (KAN). Magnetic beads were applied for separation, and gold nanoparticles (AuNPs) for signal amplification. DNA aptamers against OTC and KAN were firstly designed. After specific recognition events, the aptamer sequences were released from the surface of magnetic beads and the remaining DNA probes captured horseradish peroxidase (HRP) modified AuNPs. Subsequently, 3,3',5,5'-tetramethylbenzidine and o-phenylenediamine are catalytically oxidized by HRP, and the generated colorimetric responses can reflect the concentrations of OTC (at 370nm) and KAN (at 450nm), respectively. Experimental results demonstrate that the method is highly sensitive with the detection limit as low as 1 ag mL-1 for OTC and KAN. An extremely wide linear range (over 11 orders of magnitude) is achieved. The high selectivity is attributed to the high affinity between aptamer and the substrate. The results of real sample tests also verify that the method is promising for antibiotics analysis in the applications of food monitoring and clinical diagnosis. Graphical abstract Schematic presentation of a colorimetric assay for antibiotics based on aptamer-modified magnetic beads and horseradish peroxidase modified gold nanoparticles. Colorimetric responses result from the enzymatic oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD), respectively.

A new application of aptamer: One-step purification and immobilization of enzyme from cell lysates for biocatalysis

Aptamers are nucleic acid-based high affinity ligands that are able to capture their corresponding target through molecular recognition. In this study, several DNA aptamers with high affinity and specificity for β-glucuronidases (PGUS-E) were obtained by our modified SELEX method. Among them, Apt5 and Apt9 were selected as representatives and covalently linked to magnetic beads, respectively. The aptamer-modified magnetic beads were characterized and successfully applied to one-step purification and immobilization of PGUS-E from the complex cell lysates. By conveniently adjusting the pH and ion strength, the PGUS-E purities reached 84% for Apt5-modified beads and 88% for Apt9-modified beads. Moreover, the maximum PGUS-E capturing capacity of the Apt5 and Apt9 modified magnetic beads were found to be 31.75μg/mg and 32.95μg/mg, respectively. The immobilized PGUS-E on aptamer-based magnetic beads showed good reusability, and the conversion of glycyrrhizin still remained more than 70% after 7 cycles. In addition, the aptamer-modified beads support can be easily regenerated, and the conversion rate of glycyrrhizin (GL) was still 62% after the 7th cycle of regeneration. This investigation can be easily extended to other enzyme systems and may help open a generic route to develop a novel enzyme immobilization technology for biocatalysis based on aptamer.

Aptamer-modified magnetic beads in affinity separation of proteins.

Aptamers are valuable alternative ligands for affinity separations. Here, we describe the aptamer-based affinity separation of His-tagged proteins using an aptamer directed against the His-tag. The immobilization of the aptamer to magnetic beads is described as well as the aptamer-based purification and proper methods for the characterization of the process. Moreover, indications for the transfer of the process to other aptamers are given.

An aptamer-capture based chromogenic assay for thrombin

A simple chromogenic assay for human alpha thrombin is developed through aptamer affinity capture and a subsequent enzyme reaction. Thrombin is captured on the aptamer-modified magnetic beads, and catalyzes the conversion of chromogenic substrates to optically measured products. The measurement of the generated products by an absorbance spectrometer allows for the final quantification of thrombin. This assay shows high sensitivity by taking advantage of sample enrichment and enzyme amplification, and exhibits good specificity by involving the selective aptamer binding and the specific enzyme reaction. A concentration detection limit of 40fM can be reached when the tripeptide substrate of tosyl-Gly-Pro-Arg-p-nitroanilide is used in a 24h enzyme reaction, and the use of 2h enzyme reaction in the assay enables the detection of 400fM thrombin for a rapid analysis. This assay can be applied to detect thrombin in dilute human serum.