Aptamer Sequence Research Articles

In Situ Activation of the Receptor Aggregation for Cell Apoptosis by an AI-Au Intelligent Nanomachine via Tumor Extracellular Acidity.

Polyvalent ligand-induced cell receptor aggregation is closely related to cell behavior regulation. At present, most of the means to induce receptor aggregation rely on external stimuli such as light, heat, and magnetic fields, which may cause side effects to normal cells. How to achieve receptor aggregation on the cancer cell surface to achieve cell apoptosis selectively is still a challenge. Therefore, by taking advantage of the unique property of cancer cells' slightly acidic microenvironment, an easy-to-use apoptosis-inducing mode for the in situ activation of cell surface nucleolin clustering has been developed, which not only opened a new channel for nucleolin receptor aggregation to regulate cell function and further development but also avoided damage to normal cells, providing a new strategy for tumor treatment. Dual functional ssDNA (AS1411 aptamer and pH-responsive I-strand sequence) was modified on the surface of gold nanoparticles (AuNPs) to fabricate AI-Au intelligent nanomachines. Then, the specific binding on cancer cells and aggregation of the nucleolin receptors can be achieved via the formation of an i-Motif structure among adjacent AuNPs under the acidic microenvironment. The result showed that AI-Au nanomachines mediated nucleolin cross-linking on the cell surface, resulting in a cytotoxic effect of approximately 60%. Experiments such as calcein-AM/PI staining, nuclear dye staining, and flow cytometry demonstrated that cell apoptosis became more evident with the increase of acidity under the cell surface microenvironment. Immunofluorescence imaging further confirmed the Cyt-c/caspase-3 apoptosis pathway induced by AI-Au nanomachines. The proposed strategy used for specific cancer cell apoptosis by the in situ activation of tumor cell membrane receptor aggregation is inexpensive and simple to use, which not only provides a new means of nucleolin receptor aggregation for regulating cell function but also offers a new strategy for tumor treatment with reduced side effect to normal cells. This work is significant for comprehending the ligand-induced receptor aggregation process and can lead to the development of a promising anticancer drug.

Rational Truncation of Aptamer for Ultrasensitive Aptasensing of Chloramphenicol: Studies Using Bio-Layer Interferometry.

Aptamers are an excellent choice for the selective detection of small molecules. However, the previously reported aptamer for chloramphenicol suffers from low affinity, probably as a result of steric hindrance due to its bulky nature (80 nucleotides) leading to lower sensitivity in analytical assays. The present work was aimed at improving this binding affinity by truncating the aptamer without compromising its stability and three-dimensional folding. Shorter aptamer sequences were designed by systematically removing bases from each or both ends of the original aptamer. Thermodynamic factors were evaluated computationally to provide insight into the stability and folding patterns of the modified aptamers. Binding affinities were evaluated using bio-layer interferometry. Among the eleven sequences generated, one aptamer was selected based on its low dissociation constant, length, and regression of model fitting with association and dissociation curves. The dissociation constant could be lowered by 86.93% by truncating 30 bases from the 3' end of the previously reported aptamer. The selected aptamer was used for the detection of chloramphenicol in honey samples, based on a visible color change upon the aggregation of gold nanospheres caused by aptamer desorption. The detection limit could be reduced 32.87 times (1.673 pg mL-1) using the modified length aptamer, indicating its improved affinity as well as its suitability in real-sample analysis for the ultrasensitive detection of chloramphenicol.

Aptasensing of beta-amyloid (Aβ(1−42)) by a 3D-printed platform integrated with leaf-shaped gold nanodendrites

In this study, beta-amyloid (Aβ(1−42)), an essential biomarker for diagnosing Alzheimer’s disease (AD), was detected via an electrochemical aptasensing platform. Here, an innovative signal transducer was developed using a CO2 laser-ablated 3D-printed electrode modified with leaf-shaped gold nanodendrites (LSG NDs, mean diameter: ∼ 92 nm), which could provide an efficient interface for immobilizing aptamer strands. The modified electrode with LSG NDs exhibited an enhancement in its electrochemically active surface area about 7 times, compared with the bare electrode. This modification showed that the size, morphology, and distributions of LSG NDs in amplifying electrochemical signals might effectively provide a highly sensitive infrastructure for analyte detection. The strands of a thiol-functionalized aptamer sequence interacted with the gold surface, which created an optimized biointerface to detect Aβ(1−42) in a linear range from 0.1 pg mL−1 to 10 ng mL−1 (limit of detection (LOD): 84 fg mL−1, (S/N = 3)). The developed aptasensor confirmed satisfactory stability, desired reproducibility and regeneration, and minimal impact of interfering agents. In addition, the application of this aptasensor was monitored via an assay of spiked analyte concentrations in 20 samples, including cerebrospinal fluid (CSF) and human serum.

Customized Scaffolds for Direct Assembly of Functionalized DNA Origami.

Functional DNA origami nanoparticles (DNA-NPs) are used as nanocarriers in a variety of biomedical applications including targeted drug delivery and vaccine development. DNA-NPs can be designed into a broad range of nanoarchitectures in one, two, and three dimensions with high structural fidelity. Moreover, the addressability of the DNA-NPs enables the precise organization of functional moieties, which improves targeting, actuation, and stability. DNA-NPs are usually functionalized via chemically modified staple strands, which can be further conjugated with additional polymers and proteins for the intended application. Although this method of functionalization is extremely efficient to control the stoichiometry and organization of functional moieties, fewer than half of the permissible sites are accessible through staple modifications. In addition, DNA-NP functionalization rapidly becomes expensive when a high number of functionalizations such as fluorophores for tracking and chemical modifications for stability that do not require spatially precise organization are used. To facilitate the synthesis of functional DNA-NPs, we propose a simple and robust strategy based on an asymmetric polymerase chain reaction (aPCR) protocol that allows direct synthesis of custom-length scaffolds that can be randomly modified and/or precisely modified via sequence design. We demonstrated the potential of our strategy by producing and characterizing heavily modified scaffold strands with amine groups for dye functionalization, phosphorothioate bonds for stability, and biotin for surface immobilization. We further validated our sequence design approach for precise conjugation of biomolecules by synthetizing scaffolds including binding loops and aptamer sequences that can be used for direct hybridization of nucleic acid tagged biomolecules or binding of protein targets.

A colorimetric and photothermal dual-mode biosensing platform based on nanozyme-functionalized flower-like DNA structures for tumor-derived exosome detection.

Tumor-derived exosomes can be served as a kind of promising biomarkers for early diagnosis of cancers. Herein, a colorimetric/photothermal dual-mode exosomes sensing platform is developed for human breast cancer cell (MCF-7)-derived exosomes based on encapsulation of 3,3',5,5'-tetramethylbenzidine-loaded graphene quantum dot nanozymes (TMB-GQDzymes) into DNA flowers (DFs) via rolling circle amplification (RCA). To achieve specific detection, EpCAM aptamer for MCF-7 cell-derived exosomes is immobilized on the well plate, while the complementary sequence of another CD63 aptamer is designed into the circular template to obtain abundant capture probes. Benefitting from the dual-aptamer recognition strategy, a sandwich structure of EpCAM aptamer/exosomes/TMB-GQDzymes@DFs is formed, in which the GQDzymes can catalyze the oxidation of TMB in the presence of H2O2. The resulting products of TMB oxidation (oxTMB) can induce not only the absorption changes but also a near-infrared (NIR) laser-driven photothermal effect, achieving dual-mode detection of exosomes with the limit of detection (LOD) of 1027 particles/μL (colorimetry) and 2170 particles/μL (photothermal detection), respectively. In addition, this sensing platform has demonstrated excellent performance to well distinguish breast cancer patients from healthy individuals in serum samples analysis. Overall, the proposed dual-readout biosensor opens promising prospects for exosome detection in biological study and clinical applications.

Simple and cheap CRISPR/Cas12a biosensor based on plug-and-play of DNA aptamers for the detection of endocrine-disrupting compounds

Endocrine-disrupting compounds (EDCs) are widely distributed in the environment. Here, we present a CRISPR/Cas12a (CAS) biosensor based on DNA aptamers for point-of-care detection of EDCs. Two typical EDCs, 17β-estradiol (E2) and bisphenol A (BPA), were selected to be detected by the CAS biosensors via the plug-and-play of their DNA aptamers. The results indicated that the performance of the CAS biosensors can be well regulated by controlling the trans-cleavage activity of Cas12a on a single-stranded DNA reporter and optimizing the sequence and ratio of DNA aptamer and activator DNA. Ultimately, two reliable and specific biosensors were developed, with the linear range and limit of detection of 0.2–25 nM and 0.08 nM for E2 and of 0.1–250 nM and 0.06 nM for BPA, respectively. Compared to the existing detection methods, the CAS biosensors showed higher reliability and sensitivity with simple operation, short detection time, and no costly equipment.

Rational design enabling aptamer‐based sensors for surface‐enhanced Raman detection of small molecules

AbstractHomogeneous surface‐enhanced Raman scattering (SERS) sensors could have a significant impact in environmental analysis. The reagentless mechanism of these sensors makes them capable for in situ analysis and continuous monitoring of environmental pollutants, without having to collect environmental samples for laboratory analysis. Homogeneous SERS sensors take advantage of functional DNA strands on the surface of a SERS‐active particle. These sensors are commonly used for the detection of genomic targets. A clear path to adapt these homogeneous sensors to detect small molecules is to employ aptamer sequences. In particular, the use of duplex aptamer can make these sensors highly adaptable to different target molecules. Although there have been examples of SERS duplex aptamer sensors to detect small molecules, the design rules for this type of sensors have not been elucidated. Without design rules or a design protocol, we would not be able to fully take advantage of the adaptability of this approach to focus on new targets and applications (e.g., environmental analysis). Herein, we aim to define a protocol for the design of aptamer‐based homogeneous SERS sensors. First, we demonstrate their nonequilibrium character and the necessary length for the complementary element for the sensors to work. We demonstrate the design protocol on a well‐characterized aptamer sequence for ATP binding and then transfer the knowledge gained on an aptamer for estradiol, a common water contaminant. In this work, we establish the design protocol for a short aptamer (i.e., ATP) and analyze the issues and remedies associated with extending this protocol to longer aptamer sequences (e.g., E2).

A plug-and-play aptamer diagnostic platform based on linear dichroism spectroscopy.

A plug-and-play sandwich assay platform for the aptamer-based detection of molecular targets using linear dichroism (LD) spectroscopy as a read-out method has been demonstrated. A 21-mer DNA strand comprising the plug-and-play linker was bioconjugated onto the backbone of the filamentous bacteriophage M13, which gives a strong LD signal due to its ready alignment in linear flow. Extended DNA strands containing aptamer sequences that bind the protein thrombin, TBA and HD22, were then bound to the plug-and-play linker strand via complementary base pairing to generate aptamer-functionalised M13 bacteriophages. The secondary structure of the extended aptameric sequences required to bind to thrombin was checked using circular dichroism spectroscopy, with the binding confirmed using fluorescence anisotropy measurements. LD studies revealed that this sandwich sensor design is very effective at detecting thrombin down to pM levels, indicating the potential of this plug-and-play assay system as a new label-free homogenous detection system based on aptamer recognition.

Facile colorimetric smartphone-based biosensor for rapid detection of organophosphorus pesticides residues in environment using the aptamer-enhanced oxidase activity of octahedral Ag2O particles

The long-term and excessive use of organophosphorus pesticides (OPs) leads to hazardous residues in the environment, which threatens human health to a considerable extent. Colorimetric methods can quickly and readily identify pesticide residue, but they still have various challenges in accuracy and stability. Herein, a smartphone-assisted and non-enzymatic colorimetric biosensor was constructed for rapid monitoring of multiple OPs based on the enhanced effect of aptamer on the catalytic ability of octahedral Ag2O. It was demonstrated that the aptamer sequence could enhance the affinity of colloidal Ag2O to chromogenic substrates, and accelerate the generation of oxygen radicals such as superoxide radical (·O2−) and singlet oxygen (1O2) from dissolved oxygen, thereby significantly increasing the oxidase activity of octahedral Ag2O. The color change of the solution can be easily converted to the corresponding RGB values by a smartphone for quantitative and rapid detection of multiple OPs. Hence, the smartphone-based and visual biosensor of multiple OPs was acquired with limit of detection of 10 μg L−1, 28 μg L−1 and 40 μg L−1 for isocarbophos, profenofos and omethoate, respectively. The colorimetric biosensor also exhibited good recoveries in several environmental and biological samples, showing that it may have broad application prospects for detecting OPs residues.

Development of functional nucleic acid sensor for detection of locust pheromone 4-vinylanisole

In recent years, locust plagues have occurred frequently in the world, causing great concern. In this work, we developed a portable, stand-alone functional nucleic acid sensor to detect the locust pheromone 4-vinylanisole (4VA) to monitor the population dynamics of locusts and achieve early warning of locust disasters. A high-affinity aptamer sequence with a 4VA KD value of 269.25 ± 90.65 nM was obtained by selecting the 4VA DNA aptamer. The Apt@AuNPs@MOF (Apt: aptamer, AuNPs: gold nanoparticles, and MOF: metal–organic framework) functional nucleic acid sensor was designed on this basis, and its performance was tested. The results showed that the sensor based on the screened DNA aptamers had apparent selectivity for 4VA. At room temperature, it could respond to 1 ppm of 4VA gas within a minute. The sensitivity was 1.107 × 10−3, and the average response/recovery time was 103.1 ± 28.0 s/80.1 ± 17.4 s. The sensor was assembled with a composite adsorption tube for enrichment of gas sample, which allowed for the detection as low as 1 ppb of 4VA. It can be concluded that a gas sensor integrated with a high-affinity DNA aptamer and an interdigitated electrode automated biosensing platform provides a promising strategy on real-time monitoring the locust pheromone 4VA in ambient air for timely warning and prevention of locust plagues.

Simultaneous Dual-Sensing Platform Based on Aptamer-Functionalized DNA Hydrogels for Visual and Fluorescence Detection of Chloramphenicol and Aflatoxin M1.

In this study, a chloramphenicol and aflatoxin M1 aptamer-functionalized DNA hydrogel was designed for the simultaneous detection of chloramphenicol and aflatoxin M1 for the first time. The acrydite-modified chloramphenicol aptamer sequence was used to synthesize the DNA hydrogel and for visual detection of chloramphenicol depending on the gel-to-sol transition of the target-responsive DNA hydrogel. The DNA hydrogel formulation was set as follows: 60% of each linear polyacrylamide-DNA conjugate and 40% of acrylamide and chloramphenicol aptamer/DNA strand-1 at a molar ratio of 1:1, and the lowest concentration of chloramphenicol leading to gel dissociation was 1.0 nM at 25 °C. Furthermore, the formalized aptamer-functionalized DNA hydrogel was used to detect aflatoxin M1 by measuring the recovery of the fluorescence signal that was quenched when the FAM-labeled aflatoxin M1 aptamer and BHQ1-labeled DNA strand-2 were hybridized to form a double-stranded DNA in the network of hydrogel. The detection platform was successfully applied to the detection of chloramphenicol and aflatoxin M1, both in aqueous solution and in milk. The aptamer-functionalized DNA hydrogel had detection (LOD) and quantification limits (LOQ) for aflatoxin M1 as 1.7 and 5.2 nM, respectively. Using two aptamer sequences with high affinity and specificity, the dual-sensing platform based on the DNA hydrogel achieved higher selectivity for chloramphenicol and aflatoxin M1, which demonstrated its potential as a reliable simultaneous detection platform against two different targets for monitoring food safety.

Electrochemical Cellulase-Linked ELASA for Rapid Liquid Biopsy Testing of Serum HER-2/neu.

Non-invasive liquid biopsy assays for blood-circulating biomarkers of cancer allow both its early diagnosis and treatment monitoring. Here, we assessed serum levels of protein HER-2/neu, overexpressed in a number of aggressive cancers, by the cellulase-linked sandwich bioassay on magnetic beads. Instead of traditional antibodies we used inexpensive reporter and capture aptamer sequences, transforming the enzyme-linked immuno-sorbent assay (ELISA) into an enzyme-linked aptamer-sorbent assay (ELASA). The reporter aptamer was conjugated to cellulase, whose digestion of nitrocellulose film electrodes resulted in the electrochemical signal change. ELASA, optimized relative aptamer lengths (dimer vs monomer and trimer), and assay steps allowed 0.1 fM detection of HER-2/neu in the 10% human serum in 1.3 h. Urokinase plasminogen activator and thrombin as well as human serum albumin did not interfere, and liquid biopsy analysis of serum HER-2/neu was similarly robust but 4 times faster and 300 times cheaper than both electrochemical and optical ELISA. Simplicity and low cost of cellulase-linked ELASA makes it a perspective diagnostic tool for fast and accurate liquid biopsy detection of HER-2/neu and of other proteins for which aptamers are available.

Extracellular ATP-activated hybridization chain reaction for accurate and sensitive detection of cancer cells

Accurate and sensitive detection of cancer cells is of significant importance for early diagnosis and treatment of cancer. Here, we developed an extracellular ATP-activated hybridization chain reaction (HCR) amplification strategy to meet this purpose. This strategy relies on three DNA probes, Apt-trigger, H1-ATP aptamer duplex and hairpin H2. The Apt-trigger probe consists of two components: an aptamer sequence for specific recognition of the target cells, and a trigger sequence for the HCR assembly. The duplex structure of H1-ATP aptamer causes the toehold in hairpin H1 to be hidden, preventing the strand-displacement reaction between hairpin H1 and Apt-trigger. Upon activation with ATP, the ATP aptamer will bind to ATP to dissociate from hairpin H1, thus leading to an Apt-trigger-induced strand-displacement reaction and subsequent HCR with hairpin H2 on the target cell surface. Benefiting from aptamer recognition and ATP-activated HCR amplification, this strategy can not only perform sensitive quantitative analysis with a detection limit of 25 cells in 200 µL of binding buffer, but also show desirable specificity and accuracy for identifying target cells from control cells and mixed cell samples. Importantly, this method retains stable and good performance for target cell detection in 10% fetal bovine serum, demonstrating great potential for clinical diagnosis in complex biological matrices. Furthermore, this strategy can be adapted to detect various types of cancer cells by changing the corresponding aptamer sequence.

Pushing Adenosine and ATP SELEX for DNA Aptamers with Nanomolar Affinity.

The classical DNA aptamer for adenosine and ATP has been the most used small molecule binding aptamer for biosensing, imaging, and DNA nanotechnology. This sequence has recurred multiple times in previous aptamer selections, and all previous selections used a high concentration of ATP as the target. Herein, two separate selections were performed using adenosine and ATP as targets. By pushing the target concentrations down to the low micromolar range, two new aptamers with Kd as low as 230 nM were obtained, showing around 30-fold higher affinity compared to the classical aptamer. The classical aptamer sequence still dominated the library in the early rounds of the selections, but it was suppressed in the later rounds. The new aptamers bind to one target molecule instead of two. Mutation studies confirmed their secondary structures and specific binding. Using the deep sequencing data from the selections, long-standing questions such as the existence of one-site aptamers and mutation distribution in the classical aptamer were addressed. Comparisons were made with previously reported DNA aptamers for ATP. Finally, a strand-displacement biosensor was tested showing selectivity for adenosine and its nucleotides.

Exo-III Enzyme-Assisted Triple Cycle Signal Amplifications for Sensitive and Accurate Identification of Pathogenic Bacteria.

Early determination of infectious pathogens is vitally important to select appropriate antibiotics, and to manage nosocomial infection. Herein, we propose a target recognition triggered triple signal amplification-based approach for sensitive pathogenic bacteria detection. In the proposed approach, a double-strand DNA probe (capture probe) that is composed of an aptamer sequence and a primer sequence is designed for specific identification of target bacteria and initiation of following triple signal amplification. After recognition of target bacteria, primer sequence is released from capture probe to bind with the designed H1 probe, forming a blunt terminal in the H1 probe. Exonuclease-III (Exo-III enzyme) specifically recognizes the blunt terminal in H1 probe and degrades the sequence from 3' terminal, resulting a single-strand DNA to induce the following signal amplification. Eventually, the approach exhibits a low detection limit of 36cfu/mL with a broad dynamic range. The high selectivity endows the method a promising prospective for clinical sample analysis.

Low fouling aptasensing of rivaroxaban in real samples using poly (toluidine blue) decorated by silver nanoparticle: A new platform for the cardiovascular disease analysis

A novel ultrasensitive electrochemical aptasensor was developed for the detection of rivaroxaban (RIV). For this purpose, poly toluidine blue (P(TB)), silver nanoparticles (AgNPs), aptamer conjugated with polyethylene glycol (PEG), and ITO-PET (indium tin oxide coated polyethylene terephthalate film) electrode was utilized. In order to fabrication of the low fouling aptasensor, the surface of ITO-PET was efficiently modified by P(TB-AgNPs) to provide a suitable substrate for the stable immobilization of aptamer sequence, 5′ SH-TAG GGA AGA GAA GGA CAT ATG ATG ACT CAC AAC TGG ACG AAC GTA CTT ATC CCC CCC AAT CAC TAG TGA ATT-3′). Using ITO PET-OH-P (TB-Ag)-PEG as substrate and aptamer as bioreceptor, RIV were measured in human plasma and exhaled breath condensate samples by chronoamperometry technique with limit of detections of 6.65 and 4.13 nM, respectively. Importantly, using PEG conjugated aptamer, a low fouling aptasensor was developed for the sensitive and selective determination of RIV in spiked samples. For the first time, a novel aptasensor for determination of RIV in human plasma and exhaled breath condensate (EBC) samples was developed which is able to use for therapeutic drug monitoring (TDM) studies.

Computing the electronic circular dichroism spectrum of DNA quadruple helices of different topology: A critical test for a generalized excitonic model based on a fragment diabatization.

In this study, we exploit a recently developed fragment diabatization-based excitonic model, FrDEx, to simulate the electronic circular dichroism (ECD) spectra of three guanine-rich DNA sequences arranged in guanine quadruple helices with different topologies: thrombin binding aptamer (antiparallel), c-Myc promoter (parallel), and human telomeric sequence (3+1 hybrid). Starting from time-dependent density functional theory (TD-DFT) calculations with the M052X functional, we apply our protocol to parameterize the FrDEX Hamiltonian, which accounts for electron density overlap and includes both the coupling with charge transfer transitions and the effect of the surrounding bases on the local excitation of each chromophore. The TD-DFT/M052X spectral shapes are in good agreement with the experimental ones, the main source of discrepancy being related to the intrinsic error on the computed transition energies of guanine monomer. FrDEx spectra are fairly close to the reference TD-DFT ones, allowing a significant advance with respect to a more standard excitonic Hamiltonian. We also show that the ECD spectra are sensitive to the inclusion of the inner K cation in the calculation.

Designing Bcl-2 protein binding aptamers using an entropic fragment-based approach and in vitro validate the drug efficacy.

Antiapoptotic Bcl 2 proteins are overexpressed in cancer cells. To regulate Bcl 2 protein functions we have designed a set of nucleic acid aptamers using a patented entropic fragment-based approach (EFBA), which integrates the concept of information processing with the seed-and-grow strategy to determine the probability distribution of the nucleotide sequences that most likely interact with target structures by ruling out the ones that do not satisfy the entropic criterion. Based on molecular dynamics (MD) simulations to understand the stability of the aptamer-protein complex we finalized eight aptamer sequences and ranked them. Our discovered aptamers appeared with 9-10 nucleotides in the sequences. We then tested these aptamers to quantify their in vitro Bcl 2 protein binding ability. In this experimental validation, we applied our patented technology (US9529006B1) helping us quantify the protein-bound aptamers in actual concentration ‘micromole’ directly at the protein site. This way we quantify protein-bound aptamers in proportion to the aptamer concentrations in the protein incubating solution. Our MD simulation-based best-ranked aptamers appeared with higher in vitro protein binding potency. We then performed fluorescence quenching experiments in the Bcl 2-aptamer complex using the best aptamer, as detected in both MD simulation and in vitro binding assay-based rankings. Here we observed substantial effects on fluorescence quenching of the protein as a result of the aptamer binding. We conclude that we have found a set of aptamer candidates that may qualify as therapeutic drugs capable of regulating Bcl 2 protein functions in cancer cells and would thus favor the induction of apoptosis.

Aptasensor for the Detection of Moraxella catarrhalis Adhesin UspA2

Innovative point-of-care (PoC) diagnostic platforms are desirable to surpass the deficiencies of conventional laboratory diagnostic methods for bacterial infections and to tackle the growing antimicrobial resistance crisis. In this study, a workflow was implemented, comprising the identification of new aptamers with high affinity for the ubiquitous surface protein A2 (UspA2) of the bacterial pathogen Moraxella catarrhalis and the development of an electrochemical biosensor functionalized with the best-performing aptamer as a bioreceptor to detect UspA2. After cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) was performed, next-generation sequencing was used to sequence the final aptamer pool. The most frequent aptamer sequences were further evaluated using bioinformatic tools. The two most promising aptamer candidates, Apt1 and Apt1_RC (Apt1 reverse complement), had Kd values of 214.4 and 3.4 nM, respectively. Finally, a simple and label-free electrochemical biosensor was functionalized with Apt1_RC. The aptasensor surface modifications were confirmed by impedance spectroscopy and cyclic voltammetry. The ability to detect UspA2 was evaluated by square wave voltammetry, exhibiting a linear detection range of 4.0 × 104-7.0 × 107 CFU mL-1, a square correlation coefficient superior to 0.99 and a limit of detection of 4.0 × 104 CFU mL-1 at pH 5.0. The workflow described has the potential to be part of a sensitive PoC diagnostic platform to detect and quantify M. catarrhalis from biological samples.

A Novel Fluorescent Aptamer Sensor with DNAzyme Signal Amplification for the Detection of CEA in Blood.

Carcinoembryonic antigen (CEA) is a tumor-specific biomarker; however, its low levels in the early stages of cancer make it difficult to detect. To address the need for analysis of ultra-low-level substances, we designed and synthesized a fluorescent aptamer sensor with DNAzyme signal amplification and used it for the detection of CEA in blood. In the presence of the target protein, the aptamer sequence in the recognition probe binds to the target protein and opens the hairpin structure, hybridizes with the primer and triggers a polymerization reaction in the presence of polymerase to generate double-stranded DNA with two restriction endonuclease Nb.BbvCl cleavage sites. At the same time, the target protein is displaced and continues to bind to another recognition probe, triggering a new round of polymerization reaction, forming a cyclic signal amplification triggered by the target. The experimental results show that the blood detection with CEA has a high sensitivity and a wide detection range. The detection range: 10 fg/mL~10 ng/mL, with a detection limit of 5.2 fg/mL. In addition, the sensor can be used for the analysis of complex biological samples such as blood.