Aptamer Sequence Research Articles

Enzyme-free and highly sensitive detection of human epidermal growth factor receptor-2 based on MNAzyme signal amplification in breast cancer.

As a common cancer biomarker, human epidermal growth factor receptor-2 (HER2) is highly expressed in breast cancer. Consequently, developing a simple and accurate HER2 sensing platform is of great significance for early diagnosis and treatment of breast cancer. Herein, we developed a rapid enzyme-free fluorescent assay biosensor based on MNAzyme signal amplification for breast cancer biomarker, HER2. The MNAzyme consists of multiple parts, including complementary DNA (cDNA) and two parts of DNAzyme (partzyme A/B). Initially, cDNA is blocked by combining with the HER2 aptamer to form a double-stranded DNA. When HER2 is present, cDNA is released as a result of the binding between HER2 and its aptamer. Due to the complementary sequences among cDNA and partzyme A/B, the MNAzyme is successfully assembled to cleave the substrate, recovering the fluorescence output. The MNAzyme biosensor exhibited a low detection limit of 0.02 ng mL-1 and excellent selectivity. Furthermore, the proposed biosensor can also change the recognition element by changing the aptamer sequence to detect various biomarkers, holding great potential for cancer diagnosis and other related biomedical applications.

Towards Aptamer-Targeted Drug Delivery to Brain Tumors: The Synthesis of Ramified Conjugates of an EGFR-Specific Aptamer with MMAE on a Cathepsin B-Cleavable Linker

Background/Objectives: Targeted delivery of chemotherapeutic agents is a well-established approach to cancer therapy. Antibody–drug conjugates (ADCs) typically carry toxic payloads attached to a tumor-associated antigen-targeting IgG antibody via an enzyme-cleavable linker that releases the drug inside the cell. Aptamers are a promising alternative to antibodies in terms of antigen targeting; however, their polynucleotide nature and smaller size result in a completely different PK/PD profile compared to an IgG. This may prove advantageous: owing to their lower molecular weight, aptamer-drug conjugates may achieve better penetration of solid tumors compared to ADCs. Methods: On the way to therapeutic aptamer–drug conjugates, we aimed to develop a versatile and modular approach for the assembly of aptamer–enzymatically cleavable payload conjugates of various drug–aptamer ratios. We chose the epidermal growth factor receptor (EGFR), a transmembrane protein often overexpressed in brain tumors, as the target antigen. We used the 46 mer EGFR-targeting DNA sequence GR-20, monomethylauristatin E (MMAE) on the cathepsin-cleavable ValCit-p-aminobenzylcarbamate linker as the payload, and pentaerythritol-based tetraazide as the branching point for the straightforward synthesis of aptamer–drug conjugates by means of a stepwise Cu-catalyzed azide–alkyne cycloaddition (CuAAC) click reaction. Results: Branched aptamer conjugates of 1:3, 2:2, and 3:1 stoichiometry were synthesized and showed higher cytotoxic activity compared to a 1:1 conjugate, particularly on several glioma cell lines. Conclusions: This approach is convenient and potentially applicable to any aptamer sequence, as well as other payloads and cleavable linkers, thus paving the way for future development of aptamer–drug therapeutics by easily providing a range of branched conjugates for in vitro and in vivo testing.

Specifically Editing Cancer Sialoglycans for Enhanced In Vivo Immunotherapy through Aptamer‐Enzyme Chimeras

AbstractImmune checkpoint blockade (ICB) therapies have demonstrated remarkable clinical success in treating cancer. However, their objective response rate remains suboptimal because current therapies rely on limited immune checkpoints that fail to cover the multiple immune evasion pathways of cancer. To explore potential ICB strategies, we propose a glycoimmune checkpoint elimination (glycoICE) therapy based on targeted editing of sialoglycans on the tumor cell surface using an aptamer‐enzyme chimera (ApEC). The ApEC can be readily generated via a one‐step bioorthogonal procedure, allowing for large‐scale and uniform production. It specifically targets and desialylates cancer cells, disrupting the sialoglycan‐Siglec axis to activate immune cells and enhance immunotherapy efficacy, while its high tumor selectivity minimizes side effects from indiscriminate desialylation of normal tissues. Furthermore, the ApEC has the potential to be a versatile platform for specific editing of sialoglycans in different tumor models by adjusting the aptamer sequences to target specific protein markers. This research not only introduces a novel molecular tool for the effective editing of sialoglycans in complex environments, but also provides valuable insights for advancing DNA‐based drugs towards in vivo and clinical applications.

Towards the Development of an Optical Biosensor for the Detection of Human Blood for Forensic Analysis.

Blood is a common biological fluid in forensic investigations, offering significant evidential value. Currently employed presumptive blood tests often lack specificity and are sample destructive, which can compromise downstream analysis. Within this study, the development of an optical biosensor for detecting human red blood cells (RBCs) has been explored to address such limitations. Aptamer-based biosensors, termed aptasensors, offer a promising alternative due to their high specificity and affinity for target analytes. Aptamers are short, single-stranded DNA or RNA sequences that form stable three-dimensional structures, allowing them to bind to specific targets selectively. A nanoflare design has been employed within this work, consisting of a quenching gold nanoparticle (AuNP), DNA aptamer sequences, and complementary fluorophore-labelled flares operating through a fluorescence resonance energy transfer (FRET) mechanism. In the presence of RBCs, the aptamer-flare complex is disrupted, restoring fluorescence and indicating the presence of blood. Two aptamers, N1 and BB1, with a demonstrated binding affinity to RBCs, were selected for inclusion within the nanoflare. This study aimed to optimise three features of the design: aptamer conjugation to AuNPs, aptamer hybridisation to complementary flares, and flare displacement in the presence of RBCs. Fluorescence restoration was achieved with both the N1 and BB1 nanoflares, demonstrating the potential for a functional biosensor to be utilised within the forensic workflow. It is hoped that introducing such an aptasensor could enhance the forensic workflow. This aptasensor could replace current tests with a specific and sensitive reagent that can be used for real-time detection, improving the standard of forensic blood analysis.

Design and demonstration of a temperature-resistant aptamer structure for highly sensitive mercury ion detection with BioFETs

In this study, we developed a temperature-resistant aptamer coupled with extended gate electric double-layer Field-Effect Transistors (FETs) for highly sensitive mercury ion detection. The design of the temperature-resistant aptamer is based on the thermal and structural properties of the hairpin structure. Two aptamer sequences were designed with different melting temperatures (Tm) and compared for their sensitivity. The hairpin structure of the aptamer with a high melting temperature ensures the stable structure prior to the addition of the mercury ions, which allows the formation of thymine–mercury–thymine (T -Hg2+-T) complex in low concentrations of mercury ions. High sensitivity and low detection limit are achieved at elevated temperatures with the aptamer having a high melting temperature. The elevated temperature facilitates the reaction rate, resulting in high sensitivity and a low detection limit. The aptamer with a high melting temperature hairpin structure has shown a remarkable improvement in the limit of detection with a 4 orders of magnitude increase compared to the one with a low melting temperature. The sensor with the high melting temperature aptamer shows an extremely low detection limit of 4.68 × 10−12 M, surpassing previously reported results. The aptamer with a low melting temperature requires mercury ions to stabilize the hairpin structure by forming a T-Hg2+-T complex. The results show that if the melting temperature is lower than the ambient temperature, it is very difficult to detect low concentrations of mercury ions at the ambient temperature. The selectivity of this sequence was also tested against multiple heavy metals, including arsenic (As), lead (Pb), chromium (Cr), and cadmium (Cd) ions. This study has shown how the structural and thermal properties of the aptamer, and the ambient temperature affect the sensitivity. They are strongly correlated to the performance of the sensors and need to be considered in the design of the sequence.

Development of novel DNA aptamers and colorimetric nanozyme aptasensor for targeting multi-drug-resistant, invasive Salmonella typhimurium strain SMC25

Invasive, biofilm-forming Non-typhoidal Salmonella (iNTS), propagating through the global food and water supply chain, presents a significant risk to food safety and public health. Developing a robust detection system is crucial for enabling point-of-care, affordable, and equipment-free identification of this pathogen throughout the supply chain. In this study, we screened a novel pool of ssDNA aptamers specific to a multidrug resistant iNTS strain SMC25, previously isolated from Indian poultry products in our earlier research. Through 13 rounds of whole-cell SELEX, we identified, characterized, and selected seven full-length aptamers (ST18, ST19, ST25, ST28, ST29, ST31, and ST32). Flow cytometric analysis reveals superior binding of ST25, ST28, ST29, and ST31. These aptamers were translated onto Nanozyme-based aptasensing system for efficient, cost-effective detection of SMC25. This system harnesses the aptamer-mediated, reversible peroxidase-like activity of gold nanoparticles (GNPs) to oxidize the TMB substrate into a one-electron oxidation state, resulting in a blue-colored Diamine charge transfer complex (DCTC). The catalytic process, coupled with GNP aggregation, induces a visible color change in the test mixture from ruby-red to blue. Post-SELEX truncations identified the optimal aptamer sequence (T_ST31), which selectively detected SMC25 in water with a limit of detection (LOD) of ∼10⁴ CFU/mL. Lower concentrations (10 CFU/mL) of SMC25 could be detected after non-selective enrichment within 120 min. This research introduces a novel pool of iNTS-specific aptamers along with a cost-effective (0.25 USD per sample) solution for colorimetric detection by the naked eye.

Screening and application of aptamers as fluorescent biosensors for selective and sensitive detection of hepatocellular carcinoma and in vivo targeted delivery studies.

The incidence of primary hepatocellular carcinoma (HCC) has recently ranked fifth in the world, and the incidence rate is increasing year by year worldwide. Therefore, early diagnosis is the highest priority in the treatment of HCC. In this paper, four anti-HCC aptamers were obtained using magnetic bead SELEX technology. Among them, Apt-1 had the smallest Kd value(5.9nM) and the highest affinity. Flow cytometry results showed that the FITC-aptamers only specifically recognized HCC serum. Circular dichronism (CD) spectral characterization showed a positive peak near 275nm and a negative peak near 250nm for all aptamers, elucidating that the secondary structure formed by the candidate aptamers was a stem-loop B-DNA structure. In addition, molecular docking simulations showed that the binding of the HCC target to the candidate aptamer sequences was mainly dominated by hydrogen bonding. The results of the aptamer sensing performance analysis showed that under the optimized assay conditions, a linear relationship (ranging from 1nM to 1µM) was achieved, with a limit of detection (LOD) down to 0.75nM and a LOQ of 2.32nM. This was further validated in clinical samples, with a positive detection rate of more than 90%. Furthermore, aptamer-mediated in vivo delivery of luciferase mRNA showed that Apt-1-luciferase mRNA could be targeted to the liver and hepatic luciferase expression was significantly increased. These results demonstrate that the aptamer paves the way for clinical application, evidencing significant potential to offer reference information for early diagnosis.

Construction of Aptamer-Functionalized DNA Hydrogels for Effective Inhibition of Shiga Toxin II Toxicity.

Bacterial infections have been seriously endangering public health and life, making it imperative to explore novel anti-infection strategies for their control. Herein, we constructed a DNA hydrogel encoded with aptamers (Apt-hydrogel) to inhibit Shiga toxin II (Stx2) toxicity, thereby alleviating Escherichia coli (EHEC) infection. The Apt-hydrogel was formed by two Y-shaped DNA scaffolds through rational design, where one end of Y was encoded with an aptamer sequence targeting the B subunit of Shiga toxin II (Stx2B). The Apt-hydrogel not only retained the high affinity of the aptamer but also provided protection for the aptamer, endowing it with better stability and biocompatibility. The results from in vitro and in vivo demonstrated good mediation effects of the Apt-hydrogel on Stx2 toxicity and confirmed its excellent inhibition activity. We hypothesized that the mechanism could be attributed to the high affinity of Apt-hydrogel for Stx2B, which effectively occupies the active site of Stx2B and its receptor Gb3. This interaction enhanced steric hindrance, thereby mediating their interaction and preventing Stx2 from entering the cell to exert toxicity. We anticipate that the novel Apt-hydrogel will expand the usage of aptamers and provide a new dimension for the Apt-hydrogel as a promising blocking assistant to inhibit Shiga toxin infections via a strong steric hindrance effect.

A fluorescent platform integrated with a “one-pot” nicking endonuclease signal amplification and magnetic separation for simultaneous detection of tumor markers

Although Enzyme-linked immunosorbent assay (ELISA) has been widely used for biomedical research, simultaneous sensitive and cost-effective detection of multiple biomarkers is challenging. Herein, we proposed a “one-pot” nicking endonuclease signal amplification (NESA)-based fluorescent aptasensor for simultaneous detection of carcinoembryonic antigen (CEA) and alpha fetoprotein (AFP). Firstly, two aptamers were synchronously immobilized on the surface of magnetic nanoparticles (MNPs) by coupling with two complementary DNA (cDNA). CEA and AFP specifically recognized the aptamers and then the released cDNA (ssDNA) from the double-strands (dsDNA) triggers NESA, further breaking two detection probes which were labeled with the fluorescent dye (FAM and ROX) and its quencher (BHQ1 and BHQ2) at the same time. Then, the fluorescence signal of FAM and ROX were restored separately. The results indicated that the fluorescence intensity at the emission wavelength of 518 nm and 610 nm had a positive correlation with CEA and AFP concentrations, respectively. Under the optimum conditions, wider liner range of 1–500 ng mL−1 to CEA and 5–800 ng mL−1 to AFP of this fluorescent aptasensor were successfully obtained, achieving a detection limit of CEA and AFP were 0.7 ng mL−1 and 2 ng mL−1, respectively. Hence, it turned out that the aptasensor strategy can be a promising candidate for developing a newly fluorescence assay for the simultaneous quantitative detection of multiple tumor markers in matrix samples by changing the corresponding sequences of aptamer and fluorescent signal probe, which has great potential for the screening of early cancer.

Differential binding of curcumin with chair and basket type anti-parallel DNA G-quadruplexes

Guanine rich DNA sequences are widespread throughout the genome and they are prone to form G-quadruplex structures both in-vitro and in-vivo. Owing to their regulatory roles in important biological reactions, DNA G-quadruplexes are the prime target for the development of anti-cancer drug molecules. In the present study, we have investigated the interaction of curcumin with anti-parallel chair and basket type DNA G-quadruplexes formed by thrombin binding aptamer (TBA) and human telomeric repeat (HTG) sequences, respectively. The results obtained from spectroscopic techniques demonstrated that curcumin interacts with both the DNA G-quadruplexes through external binding mode and the estimated apparent binding constant value (Kb) was slightly higher for HTG (Kb = (4.38 ± 0.09) × 105 M−1) than that estimated for TBA (Kb = (0.62 ± 0.02) × 105 M−1) DNA G-quadruplex structures. It was also revealed that upon binding with curcumin, TBA and HTG DNA G-quadruplexes were slightly stabilized and destabilized, respectively. Circular dichroism study revealed that upon binding with curcumin the global structure of both the DNA G-quadruplexes was unaltered, whereas the local structure of HTG DNA G-quaruplex was slightly altered. Conjointly all the aforementioned spectroscopic methods emphasize that curcumin has differential interaction with anti-parallel DNA G-quadruplex structures depending on their loop orientation.

Ligand-Induced Folding in a Dopamine-Binding DNA Aptamer.

Aptamers are often employed as molecular recognition elements in the development of different types of biosensors. Many of these biosensors take advantage of the aptamer having a ligand-induced structure-formation binding mechanism. However, this binding mechanism is poorly understood. Here we use isothermal titration calorimetry, circular dichroism spectroscopy and NMR spectroscopy to study the binding and ligand-induced structural change exhibited by a dopamine-binding DNA aptamer. We analysed a series of aptamers where we shorten the terminal stem that contains the 5´and 3´termini of the aptamer sequence. All aptamers bind dopamine in an enthalpically driven process coupled with an unfavorable entropy. A general trend of the aptamer having a weaker binding affinity is observed as the terminal stem is shortened. For all aptamers studied, numerous signals appear in the imino region of the1H NMR spectrum indicating that new structure forms with ligand binding. However, it is only when this region of structure formation in the aptamer is brought close to the sensor surface that we obtain a functional electrochemical aptamer-based biosensor.

Pattern recognition-based aptasensor array for discrimination of matrix effect

Nucleic acid aptamers have attracted considerable attention in recent years, particularly in the fields of disease diagnosis and treatment and biosensing. However, complex matrix effects have severely hindered their progress towards practical application. In this study, the novel strategy of a pattern recognition-based aptasensor array was introduced for target recognition based on the predictable selectivity capability of aptamers by circumventing the challenge of the interference of the matrix effect. As one proof of concept, we chose lactoferrin as the target protein, three selected aptamers with differential affinity as its recognition probes, eleven matrix samples of milk powder as the pattern discrimination model, and AuNPs color as the response signals. In the AuNPs colorimetric arrays, the lactoferrin-added milk powder matrix induced different degrees of protection on the surface of AuNPs particles due to different degrees of binding of lactoferrin to the three aptamer sequences and therefore exhibited differentiated red-to-blue color change along with salt-induced aggregation behaviors. The difference in color responses exhibited with and without lactoferrin addition was considered as the fingerprint pattern of the lactoferrin in each milk powder matrix, followed by the supervised machine learning analysis of linear discriminant analysis (LDA) for better orthogonality to complete the identification and prediction of the unknown samples. By this strategy, eleven kinds of milk powder at the added concentration of lactoferrin (50 nmol/L) presented a distinct response pattern and have been identified and classified with accuracies of 100 % via LDA patterns (three aptamers × eleven milk powder matrices × five replicates) with three canonical factors (91.2 %, 8.58 %, and 0.22 %). Furthermore, the accuracy of 22 unknown samples was 100 %, indicating the achievement in differentiating between different milk powder matrices and the identification of each matrix. The proposed aptasensor array complements the traditional “lock-and-key” single aptasensor and opens a new direction for developing sensitive sensing array systems circumventing complex matrix effects.

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.

Development of a highly sensitive and selective electrochemical aptasensor for the detection of Staphylococcus aureus in various samples

Food poisoning caused by toxins produced by Staphylococcus aureus (S. aureus) is a significant global public health concern, impacting both developing and developed countries. One effective method for detecting S. aureus is the use of electrochemical aptasensors. This study introduces a highly sensitive and selective electrochemical aptasensor designed for detecting of S. aureus bacteria. The aptasensor incorporates ferrocene (Fc) as an electrochemical signal tag, which is covalently bonded to a zeolitic imidazolate framework-8 (ZIF-8). To enhance the performance of the working electrode, a nanocomposite of three-dimensional reduced graphene oxide and chitosan is used to create a nanomaterial film with a large surface area and excellent conductivity. The S. aureus-specific aptamer sequence is immobilized as the bio-recognition element, and cDNA-ZIF-8-Fc is hybridized with the immobilized aptamer. Differential pulse voltammograms of the modified electrode are recorded before and after the interaction between the immobilized aptamer and S. aureus. The changes in peak current serve as the analytical signal for the quantitative measurement of S. aureus. The designed aptasensor can detect S. aureus within a linear concentration range of 15.0 to 1.5 × 107 CFU mL−1, with a limit of detection (LOD) of 5.3 CFU mL−1. The results demonstrate that the aptasensor effectively quantifies S. aureus selectively in the presence of other bacterial species. Additionally, satisfactory results are obtained when measuring S. aureus in tap water and milk samples.

A liquid crystal-decorated aptasensing gadget for rapid monitoring of A549 cells: Future portable test kit for lung cancer diagnosis

BackgroundPresented here is a straightforward detection system designed to track non-small cell lung cancer (specifically A549 cells) using a combination of liquid crystals (LCs) and aptamer sequences, marking a pioneering approach in this field. A change in the alignment of LCs from perpendicular to random status by the aptamer-cell complex altered the murky polarized background of the aptasensor to multicolored. ResultsThe LC-designed aptasensor could determine A549 cancerous cells in the range of 2.0E+01−7.0E+07 cell mL−1 with a limit of detection (LOD) as low as 10 cell mL−1. Through precise quantification of A549 cells in human serum samples diluted 20 times, with recovery rates ranging from 97.59 % to 101.31 %, the suggested aptasensor proves to be a dependable method for cancer screening. Furthermore, the LC aptasensor was identified as a fast sensing array due to a 10-min incubation period for the aptamer-cell complexation. SignificanceThe LC aptasensor is label-free, operator-independent, low-cost, sensitive, and user-friendly, making it potent as a miniaturized portable sensing chip for efficient healthcare monitoring.

Single-stranded DNA regulated peroxidase mimetic activity of MCOF@AuNPs for colorimetric detection of carcinoembryonic antigen

Rapid, accurate, simple and portable monitoring of carcinoembryonic antigen (CEA) in serum is important for assessing the occurrence, development, and prognosis of cancer. In this work, we developed a colorimetric method based on the peroxidase-like activity of MCOF@AuNPs functionalized by complementary sequences of CEA-specific aptamer (MCOF@AuNPs-apt*) enhancing by G-terminated CEA-specific aptamer (G-apt). The enhancement of catalytic activity depends on the increase affinity of MCOF@AuNPs-apt*-G-apt to the TMB via promoting electron transfer. Based on this strategy, we successfully detected CEA at 0.1 ng/mL with high accuracy and anti-interference ability. Importantly, combined with the smartphone app based on the smartphone app RGB color model, the results can be visualized directly without expensive equipment, which meets the demand for point of care testing of CEA. This study also explored the enhancement of catalytic activity of nanozyme by ssDNA and the application potential in aptamer biosensor.

Perylene Diimide-based Fluorescent Aptasensor for Quantitative Analysis of Pb2+ Based on Terminal Deoxynucleotidyl Transferase-assisted Formation of Elongated Aptamer and Gold Nanoparticles.

Due to the exceedingly poisonous properties of Pb2+, it is imperative to conduct a thorough assessment of its quantity in both biological and environmental samples, as this is crucial for safeguarding public health. This study describes an economic turn-off fluorescent aptasensor for the quantitative analysis of Pb2+ employing 3,4,9,10-perylenetetracarboxylic acid diimide (PTCDI) as a cost-effective fluorophore, gold nanoparticles (AuNPs) as separating agent and an elongated aptamer as both targeting agent and PTCDI loading site. The fundamental principle of the suggested fluorescent aptasensor, which is based on PTCDI, relies on detecting variations in the fluorescence intensity of PTCDI when an elongated aptamer (as single-stranded DNA) is present or absent. The advanced aptasensor is advantageous due to the elongation of the lead aptamer sequence length induced by terminal deoxynucleotidyl transferase (TdT), resulting in enhanced sensitivity. The presence of Pb2+ and the centrifugation process causes the separation of the poly A-modified aptamer/Pb2+ conjugate from the poly T sequence. Hence, the interaction of PTCDI with the poly A moiety in the modified aptamer leads to a decrease in its fluorescence emission. The findings showcased that the fluorescent aptasensor exhibited exceptional specificity towards Pb2+ ions, while the biosensing platform accomplished an impressive detection limit of 3.7pM. Moreover, the suggested aptasensor utilizing PTCDI exhibits a commendable capability in quantitatively analyzing Pb2+ within human serum samples and mineral water.

3D-printed electrochemical cells for multi-point aptamer-based drug measurements.

Electrochemical aptamer-based (E-AB) sensors achieve detection and quantitation of biomedically relevant targets such as small molecule drugs and protein biomarkers in biological samples. E-ABs are usually fabricated on commercially available macroelectrodes which, although functional for rapid sensor prototyping, can be costly and are not compatible with the microliter sample volumes typically available in biorepositories for clinical validation studies. Seeking to develop a multi-point sensing platform for sensor validation in sample volumes characteristic of clinical studies, we report a protocol for in-house assembly of 3D-printed E-ABs. We employed a commercially available 3D stereolithographic printer (FormLabs, $5k USD) for electrochemical cell fabrication and directly embedded electrodes within the 3D-printed cell structure. This approach offers a reproducible and reusable electrode fabrication process resulting in four independent and simultaneous measurements for statistically weighted results. We demonstrate compatibility with aptamer sequences binding antibiotics and antineoplastic agents. We also demonstrate a proof-of-concept validation of serum vancomycin measurements using clinical samples. Our results demonstrate that 3D-printing can be used in conjunction with E-ABs for accessible, rapid, and statistically meaningful validation of E-AB sensors in biological matrices.

Dual-Purpose Aptamer-Based Sensors for Real-Time, Multiplexable Monitoring of Metabolites in Cell Culture Media.

The availability of high-frequency, real-time measurements of the concentrations of specific metabolites in cell culture systems will enable a deeper understanding of cellular metabolism and facilitate the application of good laboratory practice standards in cell culture protocols. However, currently available approaches to this end either are constrained to single-time-point and single-parameter measurements or are limited in the range of detectable analytes. Electrochemical aptamer-based (EAB) biosensors have demonstrated utility in real-time monitoring of analytes in vivo in blood and tissues. Here, we characterize a pH-sensing capability of EAB sensors that is independent of the specific target analyte of the aptamer sequence. We applied this dual-purpose EAB to the continuous measurement of pH and phenylalanine in several in vitro cell culture settings. The miniature EAB sensor that we developed exhibits rapid response times, good stability, high repeatability, and biologically relevant sensitivity. We also developed and characterized a leak-free reference electrode that mitigates the potential cytotoxic effects of silver ions released from conventional reference electrodes. Using the resulting dual-purpose sensor, we performed hourly measurements of pH and phenylalanine concentrations in the medium superfusing cultured epithelial tumor cell lines (A549, MDA-MB-23) and a human fibroblast cell line (MRC-5) for periods of up to 72 h. Our scalable technology may be multiplexed for high-throughput monitoring of pH and multiple analytes in support of the broad metabolic qualification of microphysiological systems.

In vitro study of a new theranostic smart niosomal nanostructure for direct delivery of docetaxel via anti-PSMA aptamer

In this study, a novel quantum dot (QD)-labeled specific anti-prostate-specific membrane antigen (PSMA) aptamer sequence was conjugated to a pH-responsive niosomal particle platform for delivery of docetaxel (DTX) components. The target cells were overexpressed PSMA. This strategy can minimize the systemic toxicity prevalent in DTX. Synthesis of pH-responsive niosomes was achieved by using thin-film hydration. The conjugation of the aptamer A10 to the niosomal particle was done via a disulfide bond. Furthermore, CdSe/ZnS QDs were fabricated using a hot-injection process, then were functionalized with mercapto propanoic acid (MPA) ligands and attached to the 3’ terminal of aptamer via an Amide bind. Moreover, several characterization analyses including dynamic light scattering (DLS), zeta potential, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM), and transmission electron microscope (TEM) were performed. Additionally, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and apoptosis assays, as well as fluorescence microscopy, were used to assess the performance of the fabricated system. The data revealed a homogenous round-shaped population of niosomes with an average size of 200 nm and a negative surface charge was synthesized successfully. The FTIR and XRD evaluations confirmed the fabrication of both QDs and niosomes and the bioconjugation processes. The drug release happened in a controlled manner with a pH-sensitivity feature. The cellular uptake of aptamer-conjugated particles enhanced and consequently caused more cytotoxicity of prostate cancer cells with overexpression of PSMA. Furthermore, the QDs provided an ability to trace the treatment and its uptake via the targeted tissue. Overall, this study contributed to the development of a low-risk, highly specific platform for the delivery of both therapeutics and imaging agents.