Aptamer Probe Research Articles

Novel Approach to Overcome Defects of Cell-SELEX in Developing Aptamers against Aspartate β-Hydroxylase.

Cell-based aptamer selection (Cell-SELEX) against predefined protein targets that benefits using the native form of the targets is the most promising approach to achieve aptamer probes capable of recognizing targets under both in vitro and in vivo conditions. The major disadvantages in Cell-SELEX are the imperfectness of the negative selection step and the lengthy procedure of selection. Here, we introduced the Counter-SELEX as part of our modified Cell-SELEX and implemented deep sequencing to overcome these shortcomings in developing aptamers against aspartate β-hydroxylase (ASPH) as a known tumor marker. In parallel with the conventional Cell-SELEX, five consecutive cycles of counter selection were accomplished using sequences bound to negative cells (the Counter-SELEX) to detect oligos that are not specific for ASPH. After high-throughput sequencing, the representative of each promising achieved family was subjected to further confirmatory analysis via flow cytometry, followed by the fluorescence immunostaining of histopathological sections. Implementing our innovative complementary method, annoying mis-selected sequences in Cell-SELEX enriched pools were effectively identified and removed. According to the affinity assay on the cells displaying ASPH, three aptamers, AP-Cell 1, AP-Cell 2, and AP-Cell 3, with Kd values of 47.51, 39.38, and 65.23 nM, respectively, were obtained, while AP-Cell 1 and 3 could then successfully spot ASPH displayed on the tissues. Our study showed that the Counter-SELEX could be considered as a complementary method for Cell-SELEX to overcome the imperfectness of the negative selection step. Moreover, high-throughput nucleotide sequencing could help to shorten the overall process.

Inside Back Cover: Aptamer Blocking Strategy Inhibits SARS‐CoV‐2 Virus Infection (Angew. Chem. Int. Ed. 18/2021)

An aptamer blocking strategy is developed by Honglin Chen, Yanling Song, Chaoyong Yang, and co-workers in their Research Article on page 10266 to inhibit SARS-CoV-2 infection. An aptamer probe was engineered to bind to the spike protein of SARS-CoV-2, which inhibits infection with the virus by blocking the interaction of the virus with ACE2 receptors on the human cell membrane.

Photoelectrochemical Aptasensors Constructed with Photosensitive PbS Quantum Dots/TiO2 Nanoparticles for Detection of Kanamycin.

Kanamycin (Kana) is widely used as a veterinary medicine and its abuse causes a serious threat to human health, raising the urgent demand for detection of residual Kana in animal-derived food with high specificity and sensitivity. Here, we developed a photoelectrochemical (PEC) biosensor for rapid quantification of Kana, with lead sulfide quantum dots/titanium dioxide nanoparticles (PbS QDs/TiO2 NPs) as a photosensitive composite, a Kana-specific DNA aptamer as a functional sensor, and ruthenium(III) hexaammine (Ru(NH3)63+) as a signal booster. To prepare the PEC aptasensor, TiO2 NPs, PbS QDs, and polyethyleneimine (PEI) were respectively used to modify the indium tin oxide electrode, and then the amine-terminated aptamer probe was connected to the PEI via glutaraldehyde. Finally, Ru(NH3)63+ was attached on the surface of the aptamer to increase the photocurrent intensity. When Kana binds competitively with Ru(NH3)63+ to the aptamer immobilized on the surface of the aptasensor, Ru(NH3)63+ will be released from the aptamer, resulting in a decrease of the photocurrent signal. This PEC aptasensor exhibits a good linear relationship between the photocurrent shift and the logarithm of Kana concentration within the range of 1.0-300.0 nmol L-1, and the detection limit is 0.161 nmol L-1. Importantly, the PEC aptasensor presented good detection selectivity owing to specific interaction with Kana and was successfully implemented to quantify Kana in honey and milk, suggesting that the PEC aptasensor has the potential of rapid detection of residual Kana in animal-derived foods.

Precise monitoring of mesenchymal stem cell homing to injured kidney with an activatable aptamer probe generated by cell-SELEX

Deficiency of precise tracking tools to monitor the migration and survival status of mesenchymal stem cells (MSCs) in the animal body has bottlenecked the clinical translation of stem cell therapies. In this study, we have for the first time successfully screened an aptamer, termed seq3, with high specificity and affinity for mouse bone marrow MSCs (mBMSCs) by MSC-based systematic evolution of ligands by exponential enrichment (MSC-SELEX), and ingeniously designed seq3-based activatable aptamer probes (AAP) for live-cell tracking. As proof of concept, real-time monitoring of transplanted mBMSCs in a mouse model of chronic kidney disease (CKD) was performed with the AAPs, and compared with Fe3O4-based magnetic resonance imaging (MRI), a widely utilized cell imaging module. It was confirmed that seq3 specifically binds to mBMSCs in the animal body and the binding activates the fluorescence of otherwise quenched dyes in AAPs, substantially minimizing the background signals originating from non-target tissues and avoiding the presence of false positive and/or false negative results occurred in MR imaging. Furthermore, the seq3-AAP-based fluorescence imaging was advantageous over Fe3O4-based MR imaging in terms of indicating survival status of the migrating and colonized mBMSCs, critical information required to evaluate the effectiveness and efficiency of cell therapy strategies. AAP fluorescence imaging results showed that the transplanted mBMSCs were well distributed in the bodies of normal mice and mainly aggregated in the livers, while they tended to migrate to injured kidneys of CKD mice and facilitated the repair of kidneys as a result. Besides cell tracking, the high specificity and affinity of seq3 to mBMSCs can be widely explored to understand the fate and biological roles of stem cells in regenerating tissues in the future.

Interrogation of Electrochemical Aptamer-Based Sensors via Peak-to-Peak Separation in Cyclic Voltammetry Improves the Temporal Stability and Batch-to-Batch Variability in Biological Fluids.

Electrochemical, aptamer-based (E-AB) sensors support continuous, real-time measurements of specific molecular targets in complex fluids such as undiluted serum. They achieve these measurements by using redox-reporter-modified, electrode-attached aptamers that undergo target binding-induced conformational changes which, in turn, change electron transfer between the reporter and the sensor surface. Traditionally, E-AB sensors are interrogated via pulse voltammetry to monitor binding-induced changes in transfer kinetics. While these pulse techniques are sensitive to changes in electron transfer, they also respond to progressive changes in the sensor surface driven by biofouling or monolayer desorption and, consequently, present a significant drift. Moreover, we have empirically observed that differential voltage pulsing can accelerate monolayer desorption from the sensor surface, presumably via field-induced actuation of aptamers. Here, in contrast, we demonstrate the potential advantages of employing cyclic voltammetry to measure electron-transfer changes directly. In our approach, the target concentration is reported via changes in the peak-to-peak separation, ΔEP, of cyclic voltammograms. Because the magnitude of ΔEP is insensitive to variations in the number of aptamer probes on the electrode, ΔEP-interrogated E-AB sensors are resistant to drift and show decreased batch-to-batch and day-to-day variability in sensor performance. Moreover, ΔEP-based measurements can also be performed in a few hundred milliseconds and are, thus, competitive with other subsecond interrogation strategies such as chronoamperometry but with the added benefit of retaining sensor capacitance information that can report on monolayer stability over time.

DNAzyme Amplified Aptasensing Platform for Ochratoxin A Detection Using a Personal Glucose Meter.

Aptamer-based sensors have emerged as a major platform for detecting small-molecular targets, because aptamers can be selected to bind these small molecules with higher affinity and selectivity than other receptors such as antibodies. However, portable, accurate, sensitive, and affordable detection of these targets remains a challenge. In this work, we developed an aptasensing platform incorporating magnetic beads and a DNAzyme for signal amplification, resulting in high sensitivity. The biosensing platform was constructed by conjugating a biotin-labeled aptamer probe of small-molecular targets such as toxins and a biotin-labeled substrate strand on magnetic beads, and the DNAzyme strand hybridized with the aptamer probe to block the substrate cleavage activity. The specific binding of the small-molecular target by the aptamer probe can replace the DNAzyme strand and then induce the hybridization between the DNAzyme strand and substrate strand, and the iterative signal amplification reaction of hydrolysis and cleavage of the substrate chain occurs in the presence of a metal ion cofactor. Using invertase to label the substrate strand, the detection of small molecules of the toxin is successfully transformed into the measurement of glucose, and the sensitive analysis of small molecules such as toxins can be realized by using the household portable glucose meter as a readout. This platform is shown to detect ochratoxin, a common toxin in food, with a linear detection range of 5 orders of magnitude, a low detection limit of 0.88 pg/mL, and good selectivity. The platform is easy to operate and can be used as a potential choice for quantitative analysis of small molecules, at home or under point-of-care settings. Moreover, by changing and designing the aptamer probe and the arm of DNAzyme strand, it can be used for the analysis of other analytes.

High-Sensitive Detection of Small-Cell Lung Cancer Cells Based on Terminal Deoxynucleotidyl Transferase-Mediated Extension Polymerization Aptamer Probe.

Small-cell lung cancer (SCLC) is characterized by early metastasis and high invasiveness, poor prognosis, and a low five-year survival rate. Therefore, the development of the effective detection of SCLC cells and imaging methods has potential significance for the prognosis and treatment of SCLC. We designed a terminal deoxynucleotidyl transferase (TdT)-mediated extension polymerization aptamer probe (denoted as TEPAP). Aptamer HCC03 was used as an element of recognizing SCLC, and it was extended as a long poly(T) tail at the 3'-hydroxyl terminus by TdT and then hybridized with short poly(A) labeled with 6-carboxyfluorescein (FAM) to construct TEPAP for the high-sensitivity detection of SCLC. The results showed that the probe could specifically recognize NCI-H446 cells. Compared with HCC03 labeled with FAM, TEPAP has demonstrated a higher fluorescence signal in recognizing NCI-H446 cells, and the fluorescence intensity of TEPAP recognizing the target cells was 10 times higher than that of nontarget cells. Flow cytometric analysis showed that the detection limit of this method was as low as 17 NCI-H446 cells in 200 μL of binding buffer. In the application of clinical cytology cell blocks, the sensitivity, specificity, and accuracy of TEPAP were 89.74, 94.44, and 91.23%, respectively. The high sensitivity and specificity of TEPAP in the application of clinical samples show that the proposed probe has great potential in the diagnosis of SCLC.

Nanomaterial and Aptamer-Based Sensing: Target Binding versus Target Adsorption Illustrated by the Detection of Adenosine and ATP on Metal Oxides and Graphene Oxide.

Target molecule-induced desorption of aptamer probes from nanomaterials has been a very popular sensing method, taking advantage of the fluorescence quenching or catalytic activity of nanomaterials for signal generation. While it is generally conceived that aptamers desorb due to binding to target molecules, in this work, we examined the effect of competitive target adsorption. From five metal oxide nanoparticles including CeO2, ZnO, NiO, Fe3O4, and TiO2, only ATP was able to induce desorption of its aptamer. Adenosine could not, even though it had an even higher affinity than ATP to the aptamer. The same conclusion was also observed with a random DNA that cannot bind ATP, indicating that the desorption of DNA was due to competitive adsorption of ATP instead of aptamer binding. On graphene oxide, however, adenosine produced slightly more aptamer desorption than ATP under most of the conditions, and this can be partially attributed to the weaker interaction of negatively charged ATP with negatively charged graphene oxide. For such surface-based biosensors, it is recommended that a nonaptamer control DNA be tested side-by-side to ensure the sensing mechanism to be related to aptamer binding instead of target adsorption.

A label-free fluorescent aptasensor based on HCR and G-quadruplex DNAzymes for the detection of prostate-specific antigen.

Prostate specific antigen (PSA) has been considered as the most potential serological biomarker for the early stage detection of prostate cancer. Here, a label-free fluorescence aptasensing strategy for detecting PSA based on hybridization chain reaction (HCR) and G-quadruplex DNAzymes has been developed. This designed strategy consists of three DNA probes, aptamer probe (AP), hairpin probe 1 (H1) and hairpin probe 2 (H2). In the presence of target PSA, the aptamer sequences in AP specifically recognized PSA to form a PSA-aptamer complex, causing an AP conformation change and thus releasing the initiator, which triggered the chain-like assembly of H1 and H2 that yielded extended nicked double-stranded DNA through HCR. Upon the addition of hemin, the G-rich segments at the end of H1 and H2 self-assembled into the peroxidase-mimicking hemin/G-quadruplex DNAzymes, which catalyzed the hydrogen peroxide-mediated oxidation of thiamine to give a fluorescence signal dependent on the concentration of PSA. Under optimal conditions, a limit of detection of 0.05 nM and a linear range from 0.1 nM to 1 nM (R2 = 0.9942) were achieved by this assay. In addition, other interfering proteins, such as IgG, AFP and CEA, did not produce any significant change in the fluorescence intensity response, indicating good selectivity of this sensor for PSA detection. Finally, this proposed aptasensor was successfully used for diluted serum samples.

Identification of a novel DNA aptamer that selectively targets lung cancer serum.

Lung cancer is the leading cause of cancer-related deaths worldwide. Early diagnosis and treatment is critical to improving the 5 year survival rate of lung cancer. The identification of new options for early-stage diagnosis and therapy of lung cancer still represents a crucial challenge. Therefore, a new diagnostic method is urgently needed. In this study, we used a new modified SELEX, called serum-SELEX, to isolate aptamers that can specifically bind lung cancer serum, without any prior knowledge of their target. Among the obtained candidate aptamer sequences, Ap-LC-19 was identified as the optimal aptamer probe with the lowest dissociation constant (Kd) value of 15 ± 8.6 nM and higher affinity assessed by qPCR. Furthermore, this molecule could be a suitable aptamer for lung cancer serum and could be used as a recognition element in aptamer-based biosensors for efficient early diagnosis of lung cancer or as an innovative tool for targeted therapy. In addition, we performed MALDI-TOF MS followed by secondary peptide sequencing MS analysis for the identification of the aptamer targeted proteins. CLEC3B could be useful biomarkers for early detection of lung cancer and in monitoring its evolution.

Multifunctional gold nanoparticle based selective detection of esophageal squamous cell carcinoma cells using resonance Rayleigh scattering assay

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in China. Early and accurate diagnosis is the key to effective treatment of this disease. In this work, a resonance Rayleigh scattering (RRS) strategy was established to determine the concentration of epidermal growth factor receptor (EGFR) over-expressed ESCC cancer cells, which based on the selective binding of the anti-EGFR aptamer (Apt) and anti-EGFR antibody (Ab) multi-functionalized gold nanoparticles (AuNPs) probe (Apt-AuNPs-Ab) to the EGFR-positive cancer cell. When the probe was mixed with the Eca109 ESCC cancer cell, a distinct RRS intensity increase was observed. The probe possesses a dynamic range from 1.0 × 102-5.0 × 105 cell·mL−1 for Eca109 cells, and the detection limit is 20 cell·mL−1. Experimental results with the A2780 ovarian cancer cells line, as well as with the Eca109 ESCC cancer cell line, clearly demonstrated that the RRS assay was sensitive and selective to Eca109 than the other cancer cell lines that express a low level of EGFR. The proposed RRS assay provided a novel platform for detection of EGFR-positive cancer cells and shown the good potential for clinical diagnostics.

Organization of Protein Tyrosine Kinase-7 on Cell Membranes Characterized by Aptamer Probe-Based STORM Imaging.

Protein tyrosine kinase-7 (PTK7), as an important membrane receptor, regulates various cellular activities, including cell polarity, movement, migration, and invasion. Although lots of research studies focused on revealing its functions from the aspect of the expression of the gene and protein are present, the relationship between the spatial distribution at the single-molecule level and the function remains unclear. Through combining aptamer probe labeling and super-resolution imaging technology, after verifying the specificity and superiority of the aptamer probe, a more significant clustering distribution of PTK7 is found on the MCF10A cell basal membrane than on the apical membrane, which is thought to be related to their specific functions on different membranes. By exploring the relationship between the assembly of PTK7 and lipid rafts, actin cytoskeleton, and carbohydrate chains on the membrane, the unique distribution of PTK7 on disparate membranes is revealed to be probably because of the varied dominant position of these three factors. These findings present the detailed spatial information of PTK7 and the related potential organization mechanism on the cell membrane, which will facilitate a better understanding of the relationship between the molecular assembly and its function, as well as the overall structure of the cell membrane.

Sensitive and label-free chemiluminescence detection of malathion using exonuclease-assisted dual signal amplification and G-quadruplex/hemin DNAzyme

Malathion is one of the most commonly used organophosphorus pesticides that can cause serious harm to the ecological environment and human health. Herein, we demonstrated a label-free chemiluminescent aptasensor for the sensitive detection of malathion based on exonuclease-assisted dual signal amplification and G-quadruplex/hemin DNAzyme. Upon the addition of malathion, the aptamer probe specifically bound to the target to form a complex malathion-S3, leaving a duplex S1-S2. The complex malathion-S3 was digested by exonuclease I and the target was released. The released target was recycled to perform exonuclease I-assisted signal amplification. Furthermore, after treatment with exonuclease III, the duplex S1-S2 was converted into the secondary target ST. The secondary target ST interacted with the hairpin H1 to form a complex H1-ST, which was further digested by exonuclease III and released the secondary target. The released secondary target was recycled to perform exonuclease III-assisted signal amplification. After complete amplification, large numbers of G-quadruplex/hemin DNAzymes were generated. Under the optimal experimental conditions, the prepared aptasensor showed an excellent linear response to malathion with a detection limit of 0.47 pM. The relative standard deviations were in the range of 4.2–6.9%. Moreover, the aptasensor was successfully applied to detect malathion in spiked food and traditional Chinese medicine samples.

(Keynote) Real-Time Biosensors for Continuous Measurements of Biomolecules in Live Subjects

A biosensor capable of continuously measuring specific molecules in vivo would provide a valuable window into patients’ health status and their response to therapeutics. Unfortunately, continuous, real-time molecular measurement is currently limited to a handful of analytes (i.e. glucose and oxygen) and these sensors cannot be generalized to measure other analytes. In this talk, we will present a biosensor technology that can be generalized to measure a wide range of biomolecules in living subjects. To achieve this, we develop synthetic antibodies (aptamers) that change its structure upon binding to its target analyte and produce an electrochemical current or emit light. Our real-time biosensor requires no exogenous reagents and can be readily reconfigured to measure different target analytes by exchanging the aptamer probes in a modular manner. Using our real-time biosensor, we demonstrate the first closed loop feedback control of drug concentration in live animals and discuss potential applications of this technology. Finally, we will discuss methods for generating the aptamer probes which are at the heart of this biosensor technology.

DNA Nanodevices for Base Excision Repair Regulates ATP In Situ Imaging and Tumor Therapy.

The design of DNA nanodevices has attracted broad attention in detecting specific targets and targeted drug delivery capacities of tumor cells. Here, we report the facile fluorometric method of dual-targeting DNA nanodevices for base excision repair (BER) regulates adenosine triphosphate (ATP) in situ imaging and tumor therapy that can counteract the mutagenic effects of uracil (U) on ATP aptamer based on the binding of U-containing damaged ATP aptamer. We prove that the DNA nanodevices not only effectively deliver the aptamer probe and tumor therapy but also able to analyze the overexpression of APE1 and uracil-DNA glycosylases (UDG) in the BER pathway via ATP in situ imaging in tumor cells. Therefore, the DNA nanodevices of the BER pathway provide the potential for tumor theranostics.

A Low Spring Constant Piezoresistive Microcantilever for Biological Reagent Detection.

This paper introduces a piezoresistive microcantilever with a low spring constant. The microcantilever was fabricated with titanium (Ti) as the piezoresistor, a low spring constant polyimide (PI) layer, and a thin silicon oxide (SiO2) layer as the top and bottom passive layers, respectively. Excellent mechanical performances with the spring constant of 0.02128 N/m and the deflection sensitivity of 1.03 × 10−7 nm−1 were obtained. The output voltage fluctuation of a Wheatstone bridge, which consists of four piezoresistive microcantilevers, is less than 3 μV@3 V in a phosphate buffered saline (PBS) environment. A microcantilever aptasensor was then developed through functionalizing the microcantilevers with a ricin aptamer probe, and detections on ricin with concentrations of 10, 20, 50 and 100 ng/mL were successfully realized. A good specificity was also confirmed by using bovine serum albumin (BSA) as a blank control. The experiment results show that the Ti and PI-based microcantilever has great prospects for ultrasensitive biochemical molecule detections with high reliability and specificity.

Aptamer-Functionalized Upconverting Nanoformulations for Light-Switching Cancer-Specific Recognition and In Situ Photodynamic-Chemo Sequential Theranostics.

Biomarker-activatable theranostic formulations offer the potential for removing specific tumors with a high diagnostic accuracy and a significant pharmacological effect. Herein, we developed a novel activatable theranostic nanoformulation UAS-PD [upconversion nanophosphor (UCNP)-aptamer/ssDNA-pyropheophorbide-a (PPA)-doxyrubicin (DOX)], which can recognize specific cancer cells with sensitivity and trigger the localized photodynamic destruction and enhanced chemotherapy. UAS-PD was constructed by the conjugation of UCNPs and aptamer probes containing the photosensitizer PPA and the chemotherapeutic drug DOX. When cancer cells are present, the UAS-PD specifically binds to PTK7, an overexpressed protein present on the surface of cancer cells, through conformational recombination of the aptamer structure and switches its upconversion luminescence from 655 to 540 nm. This long-lived ratiometric optical signal provides an ultrasensitive detection limit as low as 3.9 nM for PTK7. Changes in the conformation of UAS-PD can also induce PPA to approach UCNPs, which can produce cytotoxic singlet oxygens under near-infrared excitation to destroy the cell membrane and enhance its permeability for the simultaneously released DOX that targets cellular DNA degradation, which results in a highly effective tumor-killing effect by synergistic extra-intracellular sequential damage.

Metal-induced G-quadruplex polymorphism for ratiometric and label-free detection of lead pollution in tea

Lead pollution are critical concerns for food safety and human health. Herein, a ratiometric metal-induced G-quadruplex polymorphism was introduced to construct aptamer probes, enabling label-free and ratiometric detection of lead in tea, thus is promising for on-site detection of lead pollution. The key feature of the aptamer probe is the synergistic utilization of the dual-wavelength fluorescent signal outputs from a G-quadruplex specific dye and a DNA intercalation dye under a single-wavelength excitation, leading to a more stable and reliable recognition of Pb2+ than that of analyses based on single fluorescent reporter. The aptamer probe allowed to a mix-and-read, rapid, cost-effective detection of Pb2+ with high specificity and accuracy. Pb2+ analysis in tap water and tea exhibited good performance with recovery rates of 92.3%–109.0%. The adoption of ratiometric metal-induced G-quadruplex polymorphism would be a compelling design strategy for constructing robust aptasensor, facilitating the translation of aptamer for food safety control.

A Sensitive Fluorescent Assay for Tetracycline Detection Based on Triple-helix Aptamer Probe and Cyclodextrin Supramolecular Inclusion.

Herein, an effective pyrene excimer signaled fluorescent biosensor for the determination of tetracycline based on triple-helix aptamer probe (TAP) and supramolecular inclusion of cyclodextrin was reported. The TAP was devised containing an aptamer loop, two DNA segment stems and a triplex-forming oligonucleotide (signal probe) labeled with pyrenes at 5' and 3' ends. The presence of target could result in its binding towards aptamer with a mighty affinity, leading to a conformation change of the TAP and whereupon the release of the signal probe. This liberty of signal probe enabled the formation of pyrene excimer, generating fluorescence signals. Further, signal amplification was fulfilled through the addition of γ-cyclodextrin which could interact with pyrene dimer, thus leading to an enhanced "on-state" of the sensing ensemble. In contrast, when the target was absent, the sensing ensemble remained "off-state" because of the long distance between two pyrene molecules. When the conditions were properly optimized, the increasing signal kept a linear dependence on target concentrations ranging from 5.0nM to 100nM, and the detection limit reached as low as 1.6nM. In this way, a newly-constructed, simple, and economically affordable protocol enjoys desirable efficiency, sensitivity, specificity in biosensing. Also, its universality as another attractive behalf in assaying diverse targets was envisioned with only the need of matched aptamer replacement.

Efficient immobilization of aptamers on the layered double hydroxide nanohybrids for the electrochemical proteins detection

Despite the use of layered double hydroxides (LDH) in different electrochemical (bio)sensors, the construction of aptasensors using LDH-based surfaces was not reported to the best of our knowledge. This may be due to the lack of a suitable linker to attach aptamers to the LDH-modified surface. LDH-based aptasensors are established here as very sensitive and reliable devices in serum and cerebrospinal fluid (CSF) analysis. 5′-NH2 DNA aptamer probes were immobilized on the LDH-based surfaces in a vertical conformation without any linker materials. Due to the low electron conductivity of the LDH, carbon nanotubes (CNT) with high electronic conductivity and high surface area were combined with LDH. Thrombin was used as a model protein for aptasensing. The sensor shows a linear range of 0.005–12,000 pmol L−1 and a limit of detection of 0.1 fmol L−1. Moreover, the aptasensor was used for the sensing of thrombin in CSF and serum samples acquired from both healthy and patients with different disease.