Exponential Enrichment Research Articles

Matrix Background Screening of an ssDNA Aptamer and Its Identification Against Lactopontin

Lactopontin (LPN) is a highly phosphorylated O-glycosylated acidic protein closely associated with infant gut, brain, and immune development, and its recognition is urgent due to its rising application in fortified dairy products and infant formula. In this study, an ssDNA aptamer against LPN was obtained, among which two kinds of matrix-background-assisted systematic evolution of ligands via exponential enrichment (SELEX) approaches were performed and compared. The direct approach was to utilize the sample matrix as the mixing-incubation background between the ssDNA library and LPN that can theoretically increase screening pressure and simulate practical application scenarios. The indirect approach was to utilize a PBS buffer as a screening background and to include counter-screening steps that adopt the “sample matrix” as a whole as the counter-screening target. Their screening evolutions were monitored through qPCR assays from sequence diversity convergences of each sub-library based on the change in the proportion of hetero- and homo-duplexes from the dissociation curve and melting temperature, which were also verified from the sequence statistics of high-throughput sequencing. The common sequence of Seq.I1II3 from the two approaches was finally fished out as the aptamer through multiple analyses of combining the sequence frequency, secondary structures, homology, and binding assessments, which was demonstrated good specificity and low-nanomolar affinity by qPCR assay (KD, 5.9 nM). In addition, molecular docking and a dynamics simulation were performed for their binding site prediction and affinity confirmation. This study provides a potential identifying element and a basis for accelerating the development of methods for LPN detection in dairy products.

Design of an innovative aptasensor for the detection of chemotherapeutic drug Fludarabine phosphate

Monitoring the concentration of Fludarabine phosphate, a standard chemotherapeutic drug widely used in cancer treatment, is vital for ensuring the drug’s safety and effectiveness, tailoring treatments to individual needs, and consequently improving overall patient outcomes. Regarding the limitations of conventional techniques in terms of complexity, large time measurements, and a high cost, there is an urgent need to develop simple, rapid, and cost-effective devices. In this paper, we report the design of an aptasensor for the specific and selective detection of Fludarabine. Systematic evolution of ligands by exponential enrichment (SELEX) protocol was performed to select a specific aptamer for Fludarabine. Eleven rounds were carried out, and nine sequences were selected. Based on the dissociation constant (Kd), Ful-3, exhibiting the highest affinity (18.86 nM), was chosen and integrated into a simple electrochemical aptasensing platform for Fludarabine detection. Electrochemical results demonstrated good performance of the selected aptamer in detecting Fludarabine within the analytical range of 1 to 150 pg/mL and with LOD and LOQ in the order of 0.11 pg/mL (0.31 fM) and 0.39 pg/mL (1.06 fM), respectively. The developed platform also showed good selectivity against different analogous molecules and high applicability in serum-spiked samples, with recovery percentages ranging from 96.81 to 99.04%. Considering the encouraging results, this research presents an excellent alternative in terms of simplicity, stability, ease of use, reduction of sample volume, and low cost.

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.

In vitro selection of human cerebrospinal fluid-specific aptamers using clinical samples.

Cerebrospinal fluid (CSF) leaks may occur due to numerous etiologies and are associated with severe morbidity. Currently in the U.S., confirming the presence of a CSF leak requires protein electrophoresis testing, oftentimes involving specialized processing, and there exists no point-of-care (POC) device for CSF detection. We aimed to discover a single-stranded deoxyribonucleic acid (ssDNA) aptamer capable of selectively binding to CSF-specific biomarkers, with the future goal of developing an aptamer-based POC CSF detection device. To identify a candidate aptamer, we performed Systematic Evolution of Ligands by EXponential enrichment (SELEX) using a DNA library containing a randomized 63-nucleotide (nt) stretch flanked by 2 primer-binding sites. Quantitative polymerase chain reaction (qPCR) and fluorescence anisotropy (FA) assessed aptamer binding affinity and kinetics. Following 14 SELEX cycles, 2 dominant and functionally viable 98-nt ssDNA sequences (C2 and C3) were found. C2 and C3 demonstrated ~586x and ~82x higher affinity for CSF compared to serum, respectively. Increases in FA upon aptamer exposure to higher CSF concentrations demonstrated a K1⁄2 of 5.0% and 14.1% for C2 and C3, respectively. In vitro selection of a diverse pool of ssDNA sequences yielded 2 aptamers with high selectivity for CSF-specific biomarkers, with potential for integration into a rapid POC electrochemical diagnostic system.

AptamerRunner: An accessible aptamer structure prediction and clustering algorithm for visualization of selected aptamers

Aptamers are short single-stranded DNA or RNA molecules with high affinity and specificity for targets and are generated using the iterative Systematic Evolution of Ligands by EXponential enrichment (SELEX) process. Next-generation sequencing (NGS) revolutionized aptamer selections by allowing a more comprehensive analysis of SELEX-enriched aptamers as compared to Sanger sequencing. The current challenge with aptamer NGS datasets is identifying a diverse cohort of candidate aptamers with the highest likelihood of successful experimental validation. Herein we present AptamerRunner, an aptamer sequence and/or structure clustering algorithm that synergistically integrates computational analysis with visualization and expertise-directed decision making. The visual integration of networked aptamers with ranking data, such as fold enrichment or scoring algorithm results, represents a significant advancement over existing clustering tools by providing a natural context to depict groups of aptamers from which ranked or scored candidates can be chosen for experimental validation. The inherent flexibility, user-friendly design, and prospects for future enhancements with AptamerRunner has broad-reaching implications for aptamer researchers across a wide range of disciplines.

Selection of antibacterial aptamers against Pseudomonas plecoglossicida and analysis on their potential binding proteins

Pseudomonas plecoglossicida is one of the main pathogens causing visceral white spot disease of the large yellow croaker (Pseudosciaena crocea). Although antibiotics are used to control P. plecoglossicida infections, the long-term and large-scale use of antibiotics can lead to the development of drug-resistant bacteria as well as environmental pollution. Therefore, it is necessary to develop novel antibacterial agents to treat P. plecoglossicida infections. In this study, we first developed a two-step-centrifugation method to isolate live and dead P. plecoglossicida cells. Subsequently, we used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) screening based on inhibition rate to directly isolate antibacterial aptamers from the random library without post-processing. We isolated five antibacterial aptamers, namely B1, B2, B4, B8, and B109, which showed good inhibitory effects against P. plecoglossicida. Among these, B4 showed the highest inhibitory effect (62.40 ± 4.17 %). Further analysis revealed no positive correlation between the inhibitory effect of aptamers and their affinities with the target bacterium. The B4- and B109-binding proteins were isolated from P. plecoglossicida by magnetic separation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Mass spectrometry analysis revealed that the 26 kDa ribosomal protein L2 was the probable B4-binding protein, while the 26 kDa ribosomal protein S3 and 75 kDa ribosomal protein S1 or succinate dehydrogenase flavoprotein subunit were the probable B109-binding proteins. Structural and subcellular localization analyses of these potential B4- and B109-binding proteins were also conducted. Our findings suggest that the inhibitory activity of the antibacterial aptamers against P. plecoglossicida may be mediated via their interaction with the ribosomal proteins, which can interfere with the protein synthesis process in the bacterium, affecting its growth. These findings provide the scientific basis for the development of functional antibacterial aptamers and the elucidation of their mechanisms of action.

Identification of Podoplanin Aptamers by SELEX for Protein Detection and Inhibition of Platelet Aggregation Stimulated by C-Type Lectin-like Receptor 2.

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism for the protection of tumor cells in the bloodstream and the promotion of tumor progression and metastases. The platelet C-type lectin-like receptor 2 (CLEC-2) can bind podoplanin (PDPN) on a cancer cell surface to facilitate TCIPA. Selective blockage of PDPN-mediated platelet-tumor cell interaction is a plausible strategy for inhibiting metastases. In this study, we aimed to screen for aptamers, which are the single-stranded DNA oligonucleotides that form a specific three-dimensional structure, bind to specific molecular targets with high affinity and specificity, bind to PDPN, and interfere with PDPN/CLEC-2 interactions. The systematic evolution of ligands by exponential enrichment (SELEX) was employed to enrich aptamers that recognize PDPN. The initial characterization of ssDNA pools enriched by SELEX revealed a PDPN aptamer designated as A1 displaying parallel-type G-quadruplexes and long stem-and-loop structures and binding PDPN with a material with a dissociation constant (Kd) of 1.3 ± 1.2 nM. The A1 aptamer recognized both the native and denatured form of PDPN. Notably, the A1 aptamer was able to quantitatively detect PDPN proteins in Western blot analysis. The A1 aptamer could interfere with the interaction between PDPN and CLEC-2 and inhibit PDPN-induced platelet aggregation in a concentration-dependent manner. These findings indicated that the A1 aptamer is a candidate for the development of biosensors in detecting the levels of PDPN expression. The action by A1 aptamer could result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.

Aptamer Insights in Targeting and Designing of Therapeutic Drugs.

In the therapy of chronic and dangerous conditions like as cancer, heart disease, neurological illnesses, or retinal angiopathy, we frequently require most effective dosage schedule with adequate precision and sufficient therapeutic potential. Among these entities with the capacity to behave in a target-specific way are aptamers. Usually, they are produced by an iterative screening procedure named “Systemic Evolution of Ligands by Exponential Enrichment (SELEX)” which is used to complicate nucleic acid libraries. For several biological purposes, including targeted treatment, aptamers are potential and could be easily molded into desirable needs and also be tested in a manner that is not very extravagant or pricey. Chemical modification can be employed to enhance their pharmacological actions or prevent enzymatic degradation, hence guaranteeing their chemical integrity and bioavailability in a physiological setting. The recent advancements made in drug targeting and design with the use of aptamers are the main topic of this review. This review will highlight current developments and go over prospects and problems in the fields of aptamer-based targeted therapy, including aptamer therapeutics.

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.

DNA breathing integration with deep learning foundational model advances genome-wide binding prediction of human transcription factors.

It was previously shown that DNA breathing, thermodynamic stability, as well as transcriptional activity and transcription factor (TF) bindings are functionally correlated. To ascertain the precise relationship between TF binding and DNA breathing, we developed the multi-modal deep learning model EPBDxDNABERT-2, which is based on the Extended Peyrard-Bishop-Dauxois (EPBD) nonlinear DNA dynamics model. To train our EPBDxDNABERT-2, we used chromatin immunoprecipitation sequencing (ChIP-Seq) data comprising 690 ChIP-seq experimental results encompassing 161 distinct TFs and 91 human cell types. EPBDxDNABERT-2 significantly improves the prediction of over 660 TF-DNA, with an increase in the area under the receiver operating characteristic (AUROC) metric of up to 9.6% when compared to the baseline model that does not leverage DNA biophysical properties. We expanded our analysis to in vitro high-throughput Systematic Evolution of Ligands by Exponential enrichment (HT-SELEX) dataset of 215 TFs from 27 families, comparing EPBD with established frameworks. The integration of the DNA breathing features with DNABERT-2 foundational model, greatly enhanced TF-binding predictions. Notably, EPBDxDNABERT-2, trained on a large-scale multi-species genomes, with a cross-attention mechanism, improved predictive power shedding light on the mechanisms underlying disease-related non-coding variants discovered in genome-wide association studies.

Selection and Characterization of a DNA Aptamer Recognizing High Mobility Group Box 1 Protein (HMGB1) and Enhancing Its Pro-inflammatory Activity

Background: High mobility group box 1 (HMGB1) plays an essential role in various pathological conditions, including inflammation, fibrosis, autoimmune diseases, and carcinogenesis. The quantification of HMGB1 in body fluids holds promise for clinical applications. Objectives: This study aimed to isolate high-affinity single-stranded DNA (ssDNA) aptamers that target HMGB1. Methods: In this study, ssDNA aptamers were selected using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The affinity and specificity of the aptamers were evaluated through South-Western blot analysis, enzyme-linked aptamer sorbent assay (ELASA), and aptamer-based histochemistry staining. The impact of the aptamers on the biological activity of HMGB1 was tested in the human acute monocytic leukemia cell line, THP-1. Results: An aptamer (H-ap25, dissociation constant = 8.20 ± 0.53 nmol/L) with high affinity for the HMGB1 B box was generated. Further experiments verified that H-ap25 can be used to detect HMGB1 in South-Western blot analysis, ELASA, and aptamer-based histochemistry staining. Moreover, H-ap25 significantly augmented HMGB1-induced expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, Toll-like receptor 9 (TLR9), and activation of NF-κB in THP-1 cells. Conclusions: Our results demonstrated that H-ap25 can be used both as an enhancer of HMGB1 and as a probe in research.

PD-L1 DNA aptamers isolated from agarose-bead SELEX

Increased expression and activity of the PD-L1/PD-1 pathway suppresses the activation of cytotoxic T cells, which is vital in anti-tumour defence, allowing tumours to rise, expand and progress. Current strategies using antibodies to target PD-1/PD-L1 have been very effective in cancer therapeutics and companion diagnostics. Aptamers are a new class of molecules that offer an alternative to antibodies. Herein, the systematic evolution of ligands by exponential enrichment (SELEX) using agarose slurry beads was conducted to isolate DNA aptamers specific to recombinant human PD-L1 (rhPD-L1). Isolated aptamers were sequenced and analysed using MEGA X and structural features were examined using mFold. Three aptamer candidates (P33, P32, and P12) were selected for evaluation of binding affinity (dissociation constant, Kd) using ELONA and specificity and competitive inhibition assessment using the potentiostat-electrochemical method. Among those three, P32 displayed the highest specificity (8 nM) against PD-L1. However, P32 competes for the same binding site with the control antibody, 28–8. This study warrants further assessment of P32 aptamer as a potential, cost-effective alternative tool for targeting PD-L1.

Synthesis and Properties of α-Phosphate-Modified Nucleoside Triphosphates.

This review article is focused on the progress made in the synthesis of 5'-α-P-modified nucleoside triphosphates (α-phosphate mimetics). A variety of α-P-modified nucleoside triphosphates (NTPαXYs, Y = O, S; X = S, Se, BH3, alkyl, amine, N-alkyl, imido, or others) have been developed. There is a unique class of nucleoside triphosphate analogs with different properties. The main chemical approaches to the synthesis of NTPαXYs are analyzed and systematized here. Using the data presented here on the diversity of NTPαXYs and their synthesis protocols, it is possible to select an appropriate method for obtaining a desired α-phosphate mimetic. Triphosphates' substrate properties toward nucleic acid metabolism enzymes are highlighted too. We reviewed some of the most prominent applications of NTPαXYs including the use of modified dNTPs in studies on mechanisms of action of polymerases or in systematic evolution of ligands by exponential enrichment (SELEX). The presence of heteroatoms such as sulfur, selenium, or boron in α-phosphate makes modified triphosphates nuclease resistant. The most distinctive feature of NTPαXYs is that they can be recognized by polymerases. As a result, S-, Se-, or BH3-modified phosphate residues can be incorporated into DNA or RNA. This property has made NTPαXYs a multifunctional tool in molecular biology. This review will be of interest to synthetic chemists, biochemists, biotechnologists, or biologists engaged in basic or applied research.

Isolation and characterization of ssDNA aptamers against BipD antigen of Burkholderia pseudomallei

BackgroundMelioidosis is difficult to diagnose due to its wide range of clinical symptoms. The culture method is time-consuming and less sensitive, emphasizing the importance of rapid and accurate diagnostic tests for melioidosis. Burkholderia invasion protein D (BipD) of Burkholderia pseudomallei is a potential diagnostic biomarker. This study aimed to isolate and characterize single-stranded DNA aptamers that specifically target BipD. MethodsThe recombinant BipD protein was produced, followed by isolation of BipD-specific aptamers using Systematic Evolution of Ligands by EXponential enrichment. The binding affinity and specificity of the selected aptamers were evaluated using Enzyme-Linked Oligonucleotide Assay. ResultsThe fifth SELEX cycle showed a notable enrichment of recombinant BipD protein-specific aptamers. Sequencing analysis identified two clusters with a total of seventeen distinct aptamers. AptBipD1, AptBipD13, and AptBipD50 were chosen based on their frequency. Among them, AptBipD1 exhibited the highest binding affinity with a Kd value of 1.0 μM for the recombinant BipD protein. Furthermore, AptBipD1 showed significant specificity for B. pseudomallei compared to other tested bacteria. ConclusionAptBipD1 is a promising candidate for further development of reliable, affordable, and efficient point-of-care diagnostic tests for melioidosis.

Truncations and in silico docking to enhance the analytical response of aptamer-based biosensors

Aptamers are short oligonucleotides capable of binding specifically to various targets (i.e., small molecules, proteins, and whole cells) which have been introduced in biosensors such as in the electrochemical aptamer-based (E-AB) sensing platform. E-AB sensors are comprised of a redox-reporter-modified aptamer attached to an electrode that undergoes, upon target addition, a binding-induced change in electron transfer rates. To date, E-AB sensors have faced a limitation in the translatability of aptamers into the sensing platform presumably because sequences obtained from Systematic Evolution of Ligands by Exponential Enrichment (SELEX) are typically long (>80 nucleotides) and that obtaining structural information remains time and resource consuming. In response, we explore the utility of aptamer base truncations and in silico docking to improve their translatability into E-AB sensors. Here, we first apply this to the glucose aptamer, which we characterize in solution using NMR methods to guide design and translate truncated variants in E-AB biosensors. We further investigated the applicability of the truncation and computational approaches to four other aptamer systems (vancomycin, cocaine, methotrexate and theophylline) from which we derived functional E-AB sensors. We foresee that our strategy will increase the success rate of translating aptamers into sensing platforms to afford low-cost measurements of molecules directly in undiluted complex matrices.

Enhanced SELEX Platforms for Aptamer Selection with Improved Characteristics: A Review.

This review delves into the advancements in molecular recognition through enhanced SELEX (Systematic Evolution of Ligands by Exponential Enrichment) platforms and post-aptamer modifications. Aptamers, with their superior specificity and affinity compared to antibodies, are central to this discussion. Despite the advantages of the SELEX process-encompassing stages like ssDNA library preparation, incubation, separation, and PCR amplification-it faces challenges, such as nuclease susceptibility. To address these issues and propel aptamer technology forward, we examine next-generation SELEX platforms, including microfluidic-based SELEX, capillary electrophoresis SELEX, cell-based aptamer selection, counter-SELEX, in vivo SELEX, and high-throughput sequencing SELEX, highlighting their respective merits and innovations. Furthermore, this article underscores the significance of post-aptamer modifications, particularly chemical strategies that enhance aptamer stability, reduce renal filtration, and expand their target range, thereby broadening their utility in diagnostics, therapeutics, and nanotechnology. By synthesizing these advanced SELEX platforms and modifications, this review illuminates the dynamic progress in aptamer research and outlines the ongoing efforts to surmount existing challenges and enhance their clinical applicability, charting a path for future breakthroughs in this evolving field.

Establishment of the rapid electrochemical-SELEX monitoring system by using ACEF technique and its application to dengue virus aptasensor fabrication

Aptamers, which consist of single-stranded DNA or RNA, possess considerable potential as biomolecular recognition elements. The systematic evolution of ligands by exponential enrichment (SELEX) serves as a general method for synthesizing aptamers. However, the conventional SELEX process for monitoring aptamer-target binding is time-consuming and labor-intensive, highlighting the need for rapid monitoring and selection methods. To address this problem, this study introduces a novel approach called rapid electrochemical-SELEX (RE-SELEX) monitoring method, which significantly reduces the target-aptamer binding time from 3 h to 10 min. This process is achieved by employing an alternating current electrothermal flow (ACEF) technique. The Au micro gap-type electrode was used to RE-SELEX monitoring device. As a proof-concept of this method, the dengue virus (DENV) aptamers were synthesized. For confirming the feasibility of this DENV aptamer, we analyzed target binding of produced aptamers test including gel electrophoresis, dissociation constant analysis. Then, two aptamers were selected for constructing the rapid electrochemical DENV aptasensor. The RE biosensor was achieved by ACEF technique that reduce the target-aptamer binding within 10 min. The fabricated device can detect 37 fM of the target dengue envelope protein in diluted human serum well. Thus, the proposed method provides a reliable SELEX-monitoring method for the rapid synthesis of aptamers using a simple electrochemical platform.

Development of an Electrochemical Biosensor for GFAP with Carbon-Based Substrate for Traumatic Brain Injury

This study concentrates on creating an electrochemical biosensor designed for the real-time identification and monitoring of biomarkers, specifically Glial Fibrillary Acidis Protein (GFAP). The intended applications involve the diagnosis and prognosis of Traumatic Brain Injury (TBI). TBIs pose a significant global health challenge, impacting millions each year, and existing diagnostic methods, such as neuroimaging, face constraints related to cost, radiation exposure and a greater delay in providing a diagnosis. The suggested biosensor exploits electrochemical techniques, offering benefits such as non-invasiveness and swift results. The primary objective is to advance wearable microelectrodes for TBI detection, with an emphasis on utilizing bodily fluids like sweat as practical samples. Moreover, the biosensor's versatility makes it well-suited for various environments, including extreme conditions.The objective of this research is to enhance the precision of TBI diagnosis and prognosis, thereby ultimately improving the efficacy of point-of-care treatment. Our methodology involves the utilization of the Capture Systematic Evolution of Ligands by Exponential Enrichment (Capture-SELEX) technique to create a highly specific aptamer designed for the detection of Glial Fibrillary Acidic Protein. The biosensor we are developing will feature electrodes modified with aptamers and a carbon-based substrate, enabling continuous real-time monitoring of the target substance. The primary objective of our biosensor is to identify GFAP, thereby reducing risks associated with exposure to radiation, and healthcare costs. To ensure the long-term stability of our biosensor, our design centers around a carbon-based substrate. Carbon-based nanomaterials have garnered significant attention in biosensor applications due to their diverse range of properties, encompassing chemical stability, biocompatibility, functionalization, and biodegradability.

High-Throughput Evolution Based Near-Infrared Nanosensors with 3D Printed Spheroids for Prostate Cancer Cell Diagnosis and Therapy

Prostate cancer is the second most common cancer in men worldwide, with a mortality rate of 400,000 per year. IHC is used for diagnosis from prostate biopsy, though the precision and accuracy of the current biomarkers such as prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and alpha-methylacyl-CoA racemase (AMACR) used in IHC test remain controversial. The early detection of presence and progression of prostate cancer and recurrence are still not accurately validated by current biomarkers for prostate cancer. Therefore, many are looking for alternative specific and sensitive prostate cancer biomarkers to improve outcomes. In this study, we generated ssDNA-single walled carbon nanotubes (SWCNT) from a 1018 ssDNA library and evolved ssDNA-SWCNTs that specifically bind to the surface of prostate cancer cells by Cell-SELEX (systematic evolution of ligand by exponential enrichment) method using 3D prostate cancer cell spheroids for positive selection and fibroblasts for negative selection. ssDNA functionalities incorporated into carbon nanotubes enable the specific recognition of molecular target include biomarker proteins, small molecules, and a variety of surface proteins which are unknow as biomarkers. We use the 3D prostate cancer spheroids to more in vivo-like microenvionment that are relatively similar structures to native tissues in terms of cell-cell/cell-ECM interactions resulting in cell migration, cell proliferation, ad stem cell differentiation. It is important as screening tools to find probes that can specifically target prostate cancer cells in in vivo environment. Through sequencing and clustering of the evolved library, we verified that the selected sequence could be detected on the 3D printed spheroid from day 1 using near-infrared (nIR) imaging of SWCNTs. Additionally, we observed an increase in nIR intensity of the target on day 4 indicating the potential for diagnosing different cancer stages. Furthermore, we established that loading drugs onto ssDNA-SWCNTs resulted in cell cytotoxicity. This advanced approach holds promise for enhancing the early detection and treatment of prostate cancer, addressing a critical need in the field.