Affinity Of Aptamers Research Articles

Spatiotemporally programmed release of aptamer tethered dual angiogenic growth factors.

In tissue extracellular matrix (ECM), multiple growth factors (GFs) are sequestered through affinity interactions and released as needed by proteases, establishing spatial morphogen gradients in a time-controlled manner to guide cell behavior. Inspired by these ECM characteristics, we developed an "intelligent" biomaterial platform that spatially controls the combined bioavailability of multiple angiogenic GFs, specifically vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF-BB). Utilizing aptamer affinity interactions and complementary sequences within a GelMA matrix, our platform achieves on-demand, triggered release of individual GFs which can be "programmed" in temporally-controlled, repeatable cycles. The platform features stable incorporation of dual aptamers specific for both GFs, functional aptamer-CS molecular recognition in a 3D microenvironment with long-term stability of at least 15 days at physiological temperature, and spatially localized sequestration of individual GFs. Additionally, the system allows differential amounts of GFs to be released from the same hydrogels at different time-points, mimicking dynamic GF presentation in a 3D matrix similar to the native ECM. This flexible control over individual GF release kinetics opens new possibilities for dynamic GF presentation, with adjustable release profiles to meet the spatiotemporal needs of growing engineered tissue.

Visible light-activated signal amplification PEC aptasensor for ultrasensitive zearalenone detection based on double Z-type BiOI/g-C3N4@Au/Bi2S3 heterojunctions

Zearalenone (ZEN), also known as F-2 toxin, can enter the body through the food chain and accumulate, leading to DNA break and an abnormal number of chromosomes. Therefore, it is crucial to develop a facile and sensitive method for detecting ZEN. Herein, a visible light-activated signal amplification photoelectrochemical (PEC) aptasensor for ultrasensitive detection of ZEN has been successfully constructed, exploiting the synergistic effect of the Z-type BiOI/g-C3N4/Au heterojunctions, Bi2S3 as a signal enhancer, and the remarkable affinity and specificity of aptamers. The PEC properties and electron transfer mechanism of the double Z-type heterojunctions were thoroughly investigated. When the aptamers captured the ZEN molecules, Bi2S3 and cDNA departed from the electrode, resulting in a reduction in the photocurrent signal. Therefore, the concentration of ZEN can be quantitatively analyzed by measuring the change in the photocurrent response. The constructed PEC aptasensor exhibited exceptional sensitivity and selectivity for ZEN in the concentration range of 0.1 fg⋅mL−1 ––1.0 ng⋅mL−1, with a detection limit of 0.087 fg⋅mL−1 (S/N = 3). Finally, the PEC aptasensor was successfully applied to detect ZEN in actual coix seed samples, providing some guidance for the development of PEC aptasensors for practical medicine monitoring.

An Ultrahigh Affinity DNA Aptamer for Quinine and Its Intrinsic Fluorescence Based Label-free Detection.

Measuring quinine is critical for the detection of its overdose, understanding its pharmacological and toxicological effects, and monitoring its pollution. While a previously reported aptamer named MN4 can bind quinine, it was not selected for it, leading to compromised binding affinity and specificity. In this work, a new quinine aptamer was isolated using the library immobilization capture-SELEX technique. The Q1 aptamer has a Kd value of 10 nM determined by an isothermal titration calorimetry experiment and 45 nM in a fluorescence binding assay. A 3.5 nM quinine limit of detection was obtained based on the aptamer binding-induced quenching of the intrinsic fluorescence of quinine. A large blue shift in fluorescence was observed for quinine upon binding to Q1, whereas binding to MN4 led to a very small red shift, indicating different ways of quinine binding by these two aptamers. Q1 did not bind cocaine based on NMR spectroscopy and fluorescence assays also indicated excellent selectivity against other tested molecules. This work has supplied a high affinity aptamer for quinine that can be useful for its detection and fundamental aptamer binding studies. It also highlights the advantages of using capture-SELEX to isolate aptamers for small molecules.

Higher Affinity Enables More Accurate Detection of SARS-CoV-2 in Human Saliva Using Aptamer-Based Litmus Test.

Many aptamers have been generated by systematic evolution of ligands by exponential enrichment (SELEX) to recognize spike proteins of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2&ek), some of which have been engineered into dimeric and trimeric versions for enhanced affinity for diagnostic applications. However, no studies have been conducted to compare the utilities of monomeric, dimeric and trimeric aptamers in diagnostic assays with real clinical samples to answer the question of what levels of affinity an aptamer must have for accurate clinical diagnostics. Herein, we carried out a comparative study with two monomeric aptamers MSA1 and MSA5, one dimeric aptamer and two homotrimeric aptamers constructed with MSA1 and MSA5, with affinity varying by 1000-fold. Using a colorimetric sandwich assay to analyze 48 human saliva samples, we found that the trimeric aptamer assay (Kd≈10 pM) can identify the SARS-CoV-2 infection much more accurately than the dimeric aptamer assay (Kd≈100 pM) and monomeric aptamer assay (Kd≈10,000 pM). Based on the experimental data, we theoretically predict the levels of affinity an aptamer needs to possess to achieve 80-100 % sensitivity and 100 % specificity. The findings from this study highlight the need for deriving very high affinity aptamers to enable highly accurate detection of viral infection for future pandemics.

Higher Affinity Enables More Accurate Detection of SARS‐CoV‐2 in Human Saliva Using Aptamer‐Based Litmus Test

AbstractMany aptamers have been generated by systematic evolution of ligands by exponential enrichment (SELEX) to recognize spike proteins of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2&ek), some of which have been engineered into dimeric and trimeric versions for enhanced affinity for diagnostic applications. However, no studies have been conducted to compare the utilities of monomeric, dimeric and trimeric aptamers in diagnostic assays with real clinical samples to answer the question of what levels of affinity an aptamer must have for accurate clinical diagnostics. Herein, we carried out a comparative study with two monomeric aptamers MSA1 and MSA5, one dimeric aptamer and two homotrimeric aptamers constructed with MSA1 and MSA5, with affinity varying by 1000‐fold. Using a colorimetric sandwich assay to analyze 48 human saliva samples, we found that the trimeric aptamer assay (Kd≈10 pM) can identify the SARS‐CoV‐2 infection much more accurately than the dimeric aptamer assay (Kd≈100 pM) and monomeric aptamer assay (Kd≈10,000 pM). Based on the experimental data, we theoretically predict the levels of affinity an aptamer needs to possess to achieve 80–100 % sensitivity and 100 % specificity. The findings from this study highlight the need for deriving very high affinity aptamers to enable highly accurate detection of viral infection for future pandemics.

A highly sensitive aptamer–antibody birecognized ECL sensing platform based on the cascaded reaction between CeO2@mrGO and Co-SAC@NC for E. coli O157:H7 in untreated milk

Rapid, sensitive and specific detection of foodborne pathogens is of great significance to the early warning and prevention of foodborne diseases and environmental pollution. Here, an electrochemiluminescent (ECL) biosensor with the cascaded reaction between two nanozymes was developed for the detection of E. coli O157:H7. Cobalt single-atom catalyst (Co-SAC@NC) with oxidase (OD) -like activity, as a co-reaction accelerator of O2, catalyzed oxygen to reactive oxygen species (ROSs) and boosted ECL of luminol-O2 system. Another nanozyme CeO2@mrGO with excellent phosphatase-like activity by the introduction of Fe was use to label Ab. CeO2@mrGO captured E. coli O157:H7, and then combined with Ap on the electrode surface through the affinity of aptamer and antibody to form a sandwich structure on the gold electrode (CeO2@mrGO-Ab/E. coli O157:H7/Ap-AuE). CeO2@mrGO converted AAP into AA, which consumed ROS and quenched ECL emission of luminol-O2 system. As a consequence, this ECL sensor had a linear response range of 17–1.7 × 105 CFU/mL with LOD of 2.78 CFU/mL. This aptamer-antibody birecognized system was able to be applied to assay E. coli O157:H7 in untreated contaminated milk samples with satisfactory results.

A Label-Free Electrochemical Aptamer Sensor for Sensitive Detection of Cardiac Troponin I Based on AuNPs/PB/PS/GCE.

Cardiac troponin I (cTnI) monitoring is of great value in the clinical diagnosis of acute myocardial infarction (AMI). In this paper, a highly sensitive electrochemical aptamer sensor using polystyrene (PS) microspheres as the electrode substrate material in combination with Prussian blue (PB) and gold nanoparticles (AuNPs) was demonstrated for the sensitive and label-free determination of cTnI. PS microspheres were synthesized by emulsion polymerization and then dropped onto the glassy carbon electrode (GCE); PB and AuNPs were electrodeposited on the electrode in corresponding electrolyte solutions step by step. The PS microsphere substrate provided a large surface area for the loading mass of the biological affinity aptamers, while the PB layer improved the electrical conductivity of the modified electrode, and the electroactive AuNPs exhibited excellent catalytic performance for the subsequent electrochemical measurements. In view of the above mentioned AuNPs/PB/PS/GCE sensing platform, the fabricated label-free electrochemical aptamer sensor exhibited a wide detection range of 10 fg/mL~1.0 μg/mL and a low detection limit of 2.03 fg/mL under the optimal conditions. Furthermore, this biosensor provided an effective detection platform for the analysis of cTnI in serum samples. The introduction of this sensitive electrochemical aptamer sensor provides a reference for clinically sensitive detection of cTnI.

AptaDiff: de novo design and optimization of aptamers based on diffusion models.

Aptamers are single-stranded nucleic acid ligands, featuring high affinity and specificity to target molecules. Traditionally they are identified from large DNA/RNA libraries using $in vitro$ methods, like Systematic Evolution of Ligands by Exponential Enrichment (SELEX). However, these libraries capture only a small fraction of theoretical sequence space, and various aptamer candidates are constrained by actual sequencing capabilities from the experiment. Addressing this, we proposed AptaDiff, the first in silico aptamer design and optimization method based on the diffusion model. Our Aptadiff can generate aptamers beyond the constraints of high-throughput sequencing data, leveraging motif-dependent latent embeddings from variational autoencoder, and can optimize aptamers by affinity-guided aptamer generation according to Bayesian optimization. Comparative evaluations revealed AptaDiff's superiority over existing aptamer generation methods in terms of quality and fidelity across four high-throughput screening data targeting distinct proteins. Moreover, surface plasmon resonance experiments were conducted to validate the binding affinity of aptamers generated through Bayesian optimization for two target proteins. The results unveiled a significant boost of $87.9\%$ and $60.2\%$ in RU values, along with a 3.6-fold and 2.4-fold decrease in KD values for the respective target proteins. Notably, the optimized aptamers demonstrated superior binding affinity compared to top experimental candidates selected through SELEX, underscoring the promising outcomes of our AptaDiff in accelerating the discovery of superior aptamers.

Exploring the Lower Limit of Target Concentration in Capture-SELEX Using Guanine as a Model Target.

During an aptamer selection, using a lower target concentration may result in aptamers with a higher binding affinity. Consequently, this begs the question of whether there is a lower limit for target concentration. In this work, we conducted three aptamer selections using 5 µM, 500 nM and 50 nM guanine as the targets, respectively. Successful enrichment of the same guanine aptamers was achieved at both 5 µM and 500 nM guanine, but not with 50 nM. Using 5 µM guanine, the aptamer was enriched in eight rounds of selection, compared to that for 500 nM, which was accomplished in 17 rounds. We discuss the relation of optimal target concentration to the observed Kd value of the resulting aptamers, of which the highest affinity aptamer had a measured Kd of 200 nM. Additionally, we investigated the binding of the aptamers through mutation studies, revealing a critical cytosine. Mutating this cytosine to a thymine switched the selectivity from guanine to adenine, which is reminiscent of the guanine riboswitch. This study revealed a limit in using low target concentration, and the insights described in this article will be useful for guiding the choice of target concentration during capture-SELEX.

CRISPR-based dual-aptamer proximity ligation coupled hybridization chain reaction for precise detection of tumor extracellular vesicles and cancer diagnosis

Tumor cell-derived extracellular vesicles (TEVs) contain numerous cellular molecules and are considered potential biomarkers for non-invasive liquid biopsy. However, due to the low abundance of TEVs secreted by tumor cells and their phenotypic heterogeneity, there is a lack of sensitive and specific methods to quantify TEVs. Here, we developed a dual-aptamer proximity ligation-coupled hybridization chain reaction (HCR) method for tracing TEVs, exploiting CRISPR to achieve highly sensitive detection. Taking advantage of the high binding affinity of aptamers, the two aptamers (AptEpCAM, AptHER2) exhibited the high selectivity for TEVs recognition. HCR generated long-repeated sequence containing multiple crRNA targetable barcodes, and the signals were further amplified by CRISPR upon recognizing the HCR sequences, thereby enhancing the sensitivity. Under optimal conditions, the developed method demonstrated a favorable linear relationship in the range of 2 × 103−107 particles/μL, with a limit of detection (LOD) of 3.3 × 102 particles/μL. We directly applied our assay to clinical plasma analysis, achieving 100 % accuracy in cancer diagnosis, thus demonstrating the potential clinical applications of TEVs. Due to its simplicity and rapidity, excellent sensitivity and specificity, this method has broad applications in clinical medicine.

Fabricating Polymeric Micelles with Enrichment and Cavity Effect for In Situ Enzyme Imobilization from Natural Biosystems.

Metal-organic frameworks and hydrogen-organic frameworks (MOFs and HOFs) are attractive hosts for enzyme immobilization, but they are limited to immobilizing the purified enzymes, making industrial upscaling unattractive. Herein, aptamer-modified dual thermoresponsive polymeric micelles with switchable self-assembly and core-shell structure are constructed, which enable selective immobilization of trypsin directly from complex biological systems through a cascade operation of separation and immobilization. Their steric self-assembly provides a large amount of adsorption sites on the soluble micellar shell, resulting in high adsorption capacity and excellent selectivity. Meanwhile, their aptamer affinity ligand and cavity maintain the native conformations of trypsin and offer protective effects even in harsh conditions. The maximum adsorption capacity of the polymeric micelles for trypsin was determined to be 197 mg/g at 60 min, superior to those of MOFs and HOFs. 67.2 and 86.6% of its original activity was retained for trypsin immobilized in the cavity under strong alkaline and acidic conditions, respectively.

Design of a facile and highly sensitive electrochemical impedimetric aptasensor for detection of 17β-estradiol

In this work, a facile, rapid and label-free electrochemical (EC) impedimetric aptasensor was developed for detecting 17β-estradiol (E2) based on gold particles (Au NPs) signal amplification. The aptamer was used as the recognition and capture unit to label on the surface of Au NPs. The anti-E2 aptamer labelled Au NPs (apt-Au NPs) were then anchored on the complementary DNA-immobilized gold electrode surface by hybridization. Owing to the large surface, good biocompatibility and high conductivity, Au NPs can not only provide a wonderful microenvironment for aptamer, but also play a signal amplification role, improve analytical performance of the EC sensing platform. Due to the high affinity and specificity of aptamer to E2, the formed aptamer-E2 complexes with poor conductivity increase the electron transfer resistance (Ret). Quantitative analysis for E2 was performed based on the correlation between the change of the Ret and E2 concentrations. The developed sensor exhibited excellent analytical performance with high sensitivity, selectivity and good stability for the detection of E2. The detection limit for E2 is 0.053 pM, with a linear response ranging 0.1–200 pM. Meanwhile, its application in real environmental samples was evaluated and satisfying results were achieved.

Bimetallic loaded ZIF-8 with peroxidase-like and photothermal activities for sensitive detection and efficient elimination of Listeria monocytogenes

Listeria monocytogenes is one of the main causes of bacterial infectious diseases, posing a grave threat to food safety and global public health. Therefore, sensitive and accurate detection and efficient elimination of L. monocytogenes to prevent secondary pollution is of great significance and necessary. Herein, a nanocomposite ZIF-8@Au@Pt NPs was synthesized with excellent peroxidase-like (POD) activity, SERS property and photothermal conversion property. ZIF-8@Au@Pt NPs was further cloaked with a specific aptamer (Apt2), thus presenting precise recognition to L. monocytogenes. Magnetic beads with good separation and enrichment effect were modified with another specific aptamer (Apt1), a sandwich complex was then obtained based on the high affinity of aptamers to L. monocytogenes. The supernatant containing unreacted ZIF-8@Au@Pt NPs were capable of oxidizing colorless 3,3′,5,5′-tetramethylbenzidine (TMB) into blue oxTMB in the presence of H2O2, thus enabling colorimetric testing. Meanwhile, the generated oxTMB exhibited a distinct SERS fingerprint, realizing accurate detection of L. monocytogenes through dual-mode (colorimetric and SERS), with respective detection limits of 7 CFU/mL and 5 CFU/mL. Moreover, ZIF-8@Au@Pt NPs demonstrated exceptional photothermal conversion ability (η = 71.72 %) under a near-infrared irradiation. Approaching 100 % bactericidal effect was obtained within 2 min on the precipitates by combining its function of producing reactive oxygen species (ROS) and the near-infrared photothermal property, effectively avoiding secondary pollution. The present study presented a novel approach for realizing sensitive and accurate detection and subsequent eradication of L. monocytogenes.

Screening and Specificity Analysis of Aptamer Based on MCF‐7 Breast Cancer Cell Culture Media

AbstractBreast cancer has been the most common cancer and the most prevalent tumor in women worldwide. However, a rapid, sensitive, and painless test for breast cancer has not yet been discovered. Aptamers, as sensitive molecular recognition elements, which can specifically recognize target molecules and be used in the early diagnosis of cancer. In this study, aptamers of proteins secreted by MCF‐7 breast cancer cells were screened using Med‐SELEX technology. According to the enrichment degree and homology of aptamers, the aptamer family tree was constructed. The structure of the aptamers and the docking results with the targets were then predicted. Aptamer affinity and specificity were determined by qPCR, and the optimal aptamer AY 20 was obtained. Colloidal gold colorimetry confirmed that AY20 had high specificity for breast cancer cell culture media. Therefore, the aptamer AY20 can be used as an early diagnostic tool for breast cancer. The work provides a new idea for the rapid detection of breast cancer.

Structure-Informed Design of an Ultra Bright RNA-activated Fluorophore.

Fluorogenic RNAs such as the Mango aptamers are uniquely powerful tools for imaging RNA. A central challenge has been to develop brighter, more specific, and higher affinity aptamer-ligand systems for cellular imaging. Here, we report an ultra-bright fluorophore for the Mango II system discovered using a structure-informed, fragment-based small molecule microarray approach. The new dye, Structure informed, Array-enabled LigAnD 1 (SALAD1) exhibits 3.5-fold brighter fluorescence than TO1-Biotin and subnanomolar aptamer affinity. Improved performance comes solely from alteration of dye-RNA interactions, without alteration of the chromophore itself. Multiple high-resolution structures reveal a unique and specific binding mode for the new dye resulting from improved pocket occupancy, a more defined binding pose, and a novel bonding interaction with potassium. The dye notably improves in-cell confocal RNA imaging. This work provides both introduces a new RNA-activated fluorophore and also a powerful demonstration of how to leverage fragment-based ligand discovery against RNA targets.

Engineering of dual recognition functional aptamer-molecularly imprinted polymeric solid-phase microextraction for detecting of 17β-estradiol in meat samples

In this study, an enhanced selective recognition strategy was employed to construct a novel solid-phase microextraction fiber coating for the detection of 17β-estradiol, characterized by the combination of aptamer biorecognition and molecularly imprinted polymer recognition. Benefiting from the combination of molecularly imprinted and aptamer, aptamer-molecularly imprinted (Apt-MIP) fiber coating had synergistic recognition effect. The effects of pH, ion concentration, extraction time, desorption time and desorption solvent on the adsorption capacity of Apt-MIP were investigated. The adsorption of 17β-estradiol on Apt-MIP followed pseudo-second order kinetic model, and the Freundlich isotherm. The process was exothermic and thermodynamically spontaneous. Compared with polymers that only rely on imprinted recognition, non-imprinted recognition or aptamer affinity, Apt-MIP had the best recognition performance, which was 1.30–2.20 times that of these three materials. Furthermore, the adsorption capacity of Apt-MIP for 17β-estradiol was 885.36–1487.52 times than that of polyacrylate and polydimethylsiloxane/divinylbenzone commercial fiber coatings. Apt-MIP fiber coating had good stability and could be reused for more than 15 times. Apt-MIP solid-phase microextraction coupled with high-performance liquid chromatography was successfully applied to the determination of 17β-estradiol in pork, chicken, fish and shrimp samples, with satisfactory recoveries of 79.61 %–105.70 % and low limits of detection (0.03 μg/kg). This work provides new perspectives and strategies for sample pretreatment techniques based on molecular imprinting technology and improves analytical performance.

Portable Aptasensor Based on Parallel Rolling Circle Amplification for Tumor-Derived Exosomes Liquid Biopsy.

Here, a separation-free and label-free portable aptasensor is developed for rapid and sensitive analysis of tumor-derived exosomes (TEXs). It integrated a parallel rolling circle amplification (RCA) reaction, selective binding of metal ions or small molecules to nucleic acid-specific conformations, and a low-cost, highly sensitive handheld fluorometer. Lung cancer, for example, is targeted with two typical biomarkers (mucin 1 and programmed cell death ligand 1 (PD-L1)) on its exosomes. The affinity of aptamers to the targets modulated the amount of RCA products (T-Hg2+-T and cytosine (C)-rich single-stranded DNA), which in turn affected the fluorescence intensity of quantum dots (QDs) and methylene blue (MB). The results revealed that the limit of detection (LOD) of the handheld fluorometer for cell-derived exosomes can be as low as 30 particlesmL-1. Moreover, its specificity, sensitivity, and area under the curve (AUC) are 93% (14/15), 92% (23/25), and 0.956, as determined by the analysis of 40 clinical samples. Retesting 16 of these samples with the handheld fluorometer yielded strong concordance between the fluorometer results and those acquired from clinical computed tomography (CT) and pathology.

Ultrasensitive and Wash-Free Detection of Tumor Extracellular Vesicles by Aptamer-Proximity-Ligation-Activated Rolling Circle Amplification Coupled to Single Particle ICP-MS.

Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate measurement of TEVs presents challenges due to their low abundance and potential interference from a high number of EVs derived from normal cells. Herein, an aptamer-proximity-ligation-activated rolling circle amplification (RCA) method for EV membrane recognition, coupled with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) for the quantification of TEVs, is developed. When DNA-labeled ultrasmall gold nanoparticle (AuNP) probes bind to the long chains formed by RCA, they aggregate to form large particles. Notably, small AuNPs scarcely produce pulse signals in sp-ICP-MS, thereby detecting TEVs in a wash-free manner. By leveraging the strong binding affinity of aptamers, dual aptamers for EpCAM and PD-L1 recognition, and the sp-ICP-MS technique, this method offers remarkable sensitivity and selectivity in tracing TEVs. Under optimized conditions, the present method shows a favorable linear relationship between the pulse signal frequency of sp-ICP-MS and TEV concentration within the range of 105-107 particles/mL, along with a detection limit of 1.1 × 104 particles/mL. The pulse signals from sp-ICP-MS combined with machine learning algorithms are used to discriminate cancer patients from healthy donors with 100% accuracy. Due to its simple and fast operation and excellent sensitivity and accuracy, this approach holds significant potential for diverse applications in life sciences and personalized medicine.

Magneto-fluorescent nanobiosensor with tetrahedral framework nucleic acid-confined aptamer for trace adenosine triphosphate detection

Accurate quantitation of trace-level ATP biomarkers often suffers from interference of sample matrix and remains a challenge. Herein, a novel magnetic biological nanosensor (MBNS) integrating tetrahedral framework nucleic acid (tFNA)-confined aptamers and ultrasensitive laser-induced fluorescence (LIF) was tailored and evaluated. The fluorescent aptamer nanoprobe hybridized with FAM-labelled cDNA was particularly incorporated into the inner cavity of tFNA, enabling a dual-mode mechanism that integrates molecular size selection and aptamer affinity recognition. By this strategy, the benefits of tFNA molecular sieve, the super sensitive properties of LIF, and the specific recognition of “aptamer gene codes” were integrated, as triggered the superior response of ATP. The structures, properties and reaction mechanism of tFNA, as well as tFNA modified with aptamer probe at different sites, were characterized in detail. A high level of anti-interference was achieved, shielding against non-specific adsorption even in matrices with high protein and analogue content, 103∼105 times higher than ATP. The release of fluorescent beacon from aptamer probe was facilely driven by specific binding of ATP, and an ultra-sensitive detection of 75 pmol/L ATP was obtained without amplification. It was 2–3 orders of magnitude more sensitive than most fluorescent aptasensors. Applied to serum samples, the stability and reproducibility of the resultant aptasensor have been validated with the intra- and inter-assay RSD in 1.6–6.4 % and -1.5–8.4 %, respectively. Acceptable recoveries of 96.5–106.8 % were achieved, consistent with the LC-MS method. It was expected to open a simple and efficient protocol for highly specific and ultrasensitive ATP detection in clinical diagnostics.

Investigation of the Affinity of Aptamers for Bacteria by Surface Plasmon Resonance Imaging Using Nanosomes.

The cell-SELEX method enables efficient selection of aptamers that bind whole bacterial cells. However, after selection, it is difficult to determine their binding affinities using common screening methods because of the large size of the bacteria. Here we propose a simple surface plasmon resonance imaging method (SPRi) for aptamer characterization using bacterial membrane vesicles, called nanosomes, instead of whole cells. Nanosomes were obtained from membrane fragments after mechanical cell disruption in order to preserve the external surface epitopes of the bacterium used for their production. The study was conducted on Bacillus cereus (B. cereus), a Gram-positive bacterium commonly found in soil, rice, vegetables, and dairy products. Four aptamers and one negative control were initially grafted onto a biochip. The binding of B. cereus cells and nanosomes to immobilized aptamers was then compared. The use of nanosomes instead of cells provided a 30-fold amplification of the SPRi signal, thus allowing the selection of aptamers with higher affinities. Aptamer SP15 was found to be the most sensitive and selective for B. cereus ATCC14579 nanosomes. It was then truncated into three new sequences (SP15M, SP15S1, and SP15S2) to reduce its size while preserving the binding site. Fitting the results of the SPRi signal for B. cereus nanosomes showed a similar trend for SP15 and SP15M, and a slightly higher apparent association rate constant kon for SP15S2, which is the truncation with a high probability of a G-quadruplex structure. These observations were confirmed on nanosomes from B. cereus ATCC14579 grown in milk and from the clinical strain B. cereus J066. The developed method was validated using fluorescence microscopy on whole B. cereus cells and the SP15M aptamer labeled with a rhodamine. This study showed that nanosomes can successfully mimic the bacterial membrane with great potential for facilitating the screening of specific ligands for bacteria.