Aluminum Microcomb Electrodes on Silicon Wafer for Detecting Val66Met Polymorphism in Brain-Derived Neurotrophic Factor.

OBJECTIVE: Brain-derived neurotrophic factor (BDNF) dysregulation is widely related with various psychiatric and neurological disorders, including schizophrenia, depression, Rett syndrome, and addiction, and the available evidence suggests that BDNF is also highly correlated with Parkinson's and Alzheimer's diseases. METHODS: The BDNF target sequence was detected on a capture probe attached on aluminum microcomb electrodes on the silicon wafer surface. A capture-target-reporter sandwich-type assay was performed to enhance the detection of the BDNF target. RESULTS: The limit of detection was noticed to be 100 aM. Input of a reporter sequence at concentrations >10 aM improved the detection of the target sequence by enhancing changes in the generated currents. Control experiments with noncomplementary and single- and triple-mismatches of target and reporter sequences did not elicit changes in current levels, indicating the selective detection of the BDNF gene sequence. CONCLUSION: The above detection strategy will be useful for the detection and quantification of BDNF, thereby aiding in the provision of suitable treatments for BDNF-related disorders.

Self-assembled silver nanoparticle-DNA on a dielectrode microdevice for determination of gynecologic tumors.

Nanoscale materials have been employed in the past 2 decades in applications such as biosensing, therapeutics and medical diagnostics due to their beneficial optoelectronic properties. In recent years, silver nanoparticles (AgNPs) have gained attention due to their higher plasmon excitation efficiency than gold nanoparticles, as proved by sharper and stronger plasmon resonance peaks. The current work is focused on utilizing self-assembled DNA-AgNPs on microdevices for the detection of gynecological cancers. Human papilloma virus (HPV) mostly spreads through sexual transmittance and can cause various gynecological cancers, including cervical, ovarian and endometrial cancers. In particular, oncogene E7 from the HPV strain 16 (HPV-16 E7) is responsible for causing these cancers. In this research, the target sequence of HPV-16 E7 was detected by an AgNP-conjugated capture probe on a dielectrode sensor. The detection limit was in the range between 10 and 100 aM (by 3σ estimation). The sensitivity of the AgNP-conjugated probe was 10 aM and similar to the sensitivity of gold nanoparticle conjugation sensors, and the mismatched control DNA failed to detect the target, proving selective HPV detection. Morphological assessments on the AgNPs and the sensing surfaces by high-resolution microscopy revealed the surface arrangement. This sensing platform can be expanded to develop sensors for the detection various clinically relevant targets.

Cell-targeting aptamers act as intracellular delivery vehicles.

Aptamers are single-stranded nucleic acids or peptides identified from a randomized combinatorial library through specific interaction with the target of interest. Targets can be of any size, from small molecules to whole cells, attesting to the versatility of aptamers for binding a wide range of targets. Aptamers show drug properties that are analogous to antibodies, with high specificity and affinity to their target molecules. Aptamers can penetrate disease-causing microbial and mammalian cells. Generated aptamers that target surface biomarkers act as cell-targeting agents and intracellular delivery vehicles. Within this context, the "cell-internalizing aptamers" are widely investigated via the process of cell uptake with selective binding during in vivo systematic evolution of ligands by exponential enrichment (SELEX) or by cell-internalization SELEX, which targets cell surface antigens to be receptors. These internalizing aptamers are highly preferable for the localization and functional analyses of multiple targets. In this overview, we discuss the ways by which internalizing aptamers are generated and their successful applications. Furthermore, theranostic approaches featuring cell-internalized aptamers are discussed with the purpose of analyzing and diagnosing disease-causing pathogens.

Co-immobilized poly(ethylene glycol)-block-polyamines promote sensitivity and restrict biofouling on gold sensor surface for detecting factor IX in human plasma.

In order to detect an extremely low amount of human coagulation factor IX (FIX), poly(ethylene glycol) (PEG)/aptamer co-immobilized surface was constructed using original PEG-polyamine surface modification agents on surface plasmon resonance (SPR) sensor chip. Initially, a gold (Au) sensor chip of SPR was modified using poly(ethylene glycol)-b-poly[2-(N,N-dimethylamino)ethyl methacrylate] (PEG-b-PAMA) followed by treatment with SH-dT20 and was duplexed with anti-FIX aptamer extended using A24. Furthermore, the co-immobilization of pentaethylenehexamine-terminated poly(ethylene glycol) (N6-PEG) on the sensing surface completely quenched bio-fouling. On this dual tethered PEG-surface, we determined that the dissociation constant for FIX-aptamer interaction was 37 ± 10 pM, and the sensitivity of detection could reach up to 800 fM on using aptamer-FIX-antibody sandwich pattern detected by gold nanoparticle-conjugated anti-mouse antibody. We could detect FIX in the presence of abundant albumin. Furthermore, to mimic the actual detection of FIX in clinical samples, we demonstrated our experimental results with human blood plasma instead of FIX. Higher-sensitivity was attained because of dual polymers immobilized on Au surface, and this can emerge as a common strategy for any aptamer-protein interactions. The selective binding of aptamer in human blood plasma described here indicates the suitability of the present strategy for detection in clinically relevant samples.

Zeolite-iron oxide integrated interdigitated electrode sensor for diagnosing cervical cancer.

Cervical cancer is caused by changes in the cervix that lead to precancerous cells and eventually progress to cancer. Human papillomavirus (HPV) infections are the primary cause of cervical cancer. Early detection of HPV is crucial in preventing cervical cancer, and regular screening for HPV infection can identify cell changes before they develop into cancer. While Pap smear tests are reliable for cervical cancer screening, they are critical, expensive, and labor-intensive. Therefore, researchers are focusing on identifying blood-based biomarkers using biosensors for cervical cancer screening. HPV strains 16, 45, and 18 are common culprits in cervical cancer. This study aimed to develop an HPV-16 DNA biosensor on a zeolite-iron oxide (zeolite-IO) modified interdigitated electrode (IDE) sensor. The DNA probe was immobilized on the IDE through amine-modified zeolite-IO, enhancing the hybridization of the target and DNA probe. The detection limit of the DNA-DNA duplex was found to be 7.5 pM with an R2 value of 0.9868. Additionally, control experiments with single and triple mismatched sequences showed no increase in current responses, and the identification of target DNA in a serum-spiked sample indicated specific and selective target identification.

Generation of anti-influenza aptamers using the systematic evolution of ligands by exponential enrichment for sensing applications.

The systematic evolution of ligands by exponential enrichment (SELEX) is a selection process for identifying high-affinity selective molecules from a randomized combinatorial nucleic acid library against a wide range of target molecules. Using a pool of N25 RNA molecules, the SELEX process was performed against two targets from influenza viruses, namely, intact influenza B/Tokio/53/99 and hemagglutinin of infuluenza B Jilin/20/2003. The selection processes were evaluated by surface plasmon fluorescence spectroscopy (SPFS), and the result was compared to that obtained by a conventional radioisotope method. Clear discrimination among different selection cycles was displayed by SPFS, indicating that this method can be used as an alternative method of radioisotope labeling. The dissociation constant of the selected aptamers against the targets was in the low nanomolar range. The sensitivity of the selected aptamer against intact influenza B/Tokio/53/99 to detect the influenza virus was the low ng/mL level, an approximately 250-fold higher sensitivity than that of the commercially obtained antibody. The target binding sites on the aptamer were predicted by mapping analyses. The selected aptamer could discriminate other influenza strains, and the sensitivity of the selected aptamer was further confirmed by gold-nanoparticle-based sensing on a waveguide-mode sensor. This finding demonstrates that the selected aptamer would be useful for detecting influenza viruses at an early stage of infection and for the purpose of influenza surveillance.

A high-performance waveguide-mode biosensor for detection of factor IX using PEG-based blocking agents to suppress non-specific binding and improve sensitivity.

An evanescent-field-coupled waveguide-mode (EFC-WM) sensor utilizes monolithic SiO2/Si/SiO2 sensing plates having a multilayered structure and is used to evaluate a blocking agent comprising poly(ethylene glycol)-based block copolymers. Factor IX (FIX) protein was detected using its aptamer, viz. FIX was immobilized on a glutaraldehyde-modified silica surface, and then treated with a biotinylated aptamer. The quantitative analysis of FIX was carried out using streptavidin-conjugated gold nanoparticles (SA-GNPs). The blocking polymer, poly(ethylene glycol)-b-poly(acrylic acid) (PEG-b-PAAc), was found to mask unreacted amine and glutaraldehyde (Glu) moieties on the SiO2 surface, and it completely prevented the non-specific binding of SA-GNPs. By exploiting the strong blocking effect of PEG-b-PAAc, we achieved high ligand-analyte interaction sensitivity (sensitive down to 100 pM). To improve the sensitivity further, we also used pentaethylenehexamine-terminated PEG (N6-PEG) on GNPs. The improvement in sensitivity was found to be 1000-fold (to 100 fM), which was substantiated by the observation of higher numbers of GNPs on the sensing surface in the results of the scanning electron microscopic examination. Based on the competition assay of free biotin premixed with SA-GNPs, it was concluded that some active biotin-binding sites on the streptavidin were blocked by N6-PEG, which improved the binding ability to the biotinylated sensing surface. An optimum number of binding sites on the SA-GNPs might improve their binding affinity. The strategy shown with dual polymers, viz. blocking of the sensor chip surface and coating of SA-GNPs, is recommended for developing sensors with higher sensitivity and reliability. Selective binding of the aptamer to a very small amount of FIX in the mixed sample containing FXIa and FVIIa, or albumin, makes this the optimal strategy for detecting a FIX deficiency in human blood samples.

Carbon Material Hybrid Construction on an Aptasensor for Monitoring Surgical Tumors.

Carcinoembryonic antigen (CEA) is a glycoprotein, one of the common tumor biomarkers, found at low levels in body fluids. Generally, overexpression of CEA is found in various cancers, including ovarian, breast, lung, colorectal, gastric, and pancreatic cancers. Since CEA is an important tumor biomarker, the quantification of CEA is helpful for diagnosing cancer, monitoring tumor progression, and the follow-up treatment. This research develops a highly sensitive sandwich aptasensor for CEA identification on an interdigitated electrode sensor. Carbon-based material was used to attach a higher anti-CEA capture aptamer onto the sensor surface through a chemical linker, and then, CEA was quantified by the aptamer. Furthermore, CEA-spiked serum was tested by using the immobilized aptamer, which was found to not affect the target validation. The limit of detection for CEA in PBS and serum is calculated from a linear regression graph to be 0.5 ng/mL with R 2 values of 0.9593 and 0.9657, respectively, over a linear range from 0.5 to 500 ng/mL. This CEA quantification by the aptasensor can help diagnose various surgical tumors and monitor their progression.

Non-protein coding RNA sequences mediate specific colorimetric detection of Staphylococcus aureus on unmodified gold nanoparticles.

Nonprotein coding RNA (npcRNA) is a transcribed gene sequence that is not able to translate into protein, yet it executes a specific function in modulation and regulation mechanisms. As npcRNA is highly resistant to the mutation, the Sau-02 npcRNA gene and its probe oligonucleotide, which are specifically present in Staphylococcus aureus and in methicillin-resistant S. aureus only, used to develop a highly specific and sensitive colorimetric assay on unmodified gold nanoparticles (AuNPs). Hybridization between the npcRNA Sau-02 gene sequences was detected through noncrosslinking AuNP aggregation in salt solution in the presence of probe-target gene sequences. AuNPs of 10 and 15 nm in sizes with monovalent ion salt (NaCl) solution were optimized as the ideal tool for investigating the stability of AuNPs upon the addition of gene sequences. The state dispersed and aggregated forms of 10 nm AuNPs with the presented colorimetric assay were justified through field emission scanning electron microscopy and atomic force microscopy. The particle distribution of two different AuNP states was evaluated through particle distribution analysis. The lowest detection amount of S. aureus npcRNA from the colorimetric assay performed was 6 pg/µL, as the color of AuNPs turned blue with the presence of probe oligonucleotides and target gene sequences.

Graphene oxide-gold nanoparticle-aptamer complexed probe for detecting amyloid beta oligomer by ELISA-based immunoassay.

Highly sensitive and easy detection method for Alzheimer's disease (AD) with a suitable biomarker is mandatory for preventing the factors resulting from AD. This research reports a modified ELISA with graphene for the detection of AD biomarker amyloid beta (Aß) oligomer. Gold nanoparticle (AuNP) conjugated aptamer was used as the capture probe and attached on ELISA-graphene oxide surface through the amine linker. Antibody was used as the detection molecule to reach the maximum detection of Aß oligomer. Suitable level of APTMS (2%), size of AuNP (30 nm) and aptamer concentration (2 µM) were optimized. This sandwich pattern of aptamer-Aß oligomer-antibody helps to reach the detection at 50 pM on the optimized ELISA surface and the control experiments in the absence of Aß oligomer or anti-Aß oligomer antibody did not show the significant optical detection at 492 nm, indicting the specific detection. Further, Aß oligomer spiked artificial cerebrospinal fluid did not interfere the detection of Aß oligomer, confirming the selective detection. This new and modified ELISA surface helps to reach the lower detection of Aß oligomer and diagnose AD.

Alzheimer's Disease Determination by a Dual Probe on Gold Nanourchins and Nanohorn Hybrids.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative chronic disorder that causes dementia and problems in thinking, cognitive impairment and behavioral changes. Amyloid-beta (Aß) is a peptide involved in AD progression, and a high level of Aß is highly correlated with severe AD. Identifying and quantifying Aß levels helps in the early treatment of AD and reduces the factors associated with AD. MATERIALS AND METHODS: This research introduced a dual probe detection system involving aptamers and antibodies to identify Aß. Aptamers and antibodies were attached to the gold (Au) urchin and hybrid on the carbon nanohorn-modified surface. The nanohorn was immobilized on the sensor surface by using an amine linker, and then a Au urchin dual probe was immobilized. RESULTS: This dual probe-modified surface enhanced the current flow during Aß detection compared with the surface with antibody as the probe. This dual probe interacted with higher numbers of Aß peptides and reached the detection limit at 10 fM with R2=0.992. Furthermore, control experiments with nonimmune antibodies, complementary aptamer sequences and control proteins did not display the current responses, indicating the specific detection of Aß. CONCLUSION: Aß-spiked artificial cerebrospinal fluid showed a similar response to current changes, confirming the selective identification of Aß.

Electroanalysis on an Interdigitated Electrode for High-Affinity Cardiac Troponin I Biomarker Detection by Aptamer-Gold Conjugates.

The primary reasons for myocardial infarction (MI) are pericarditis, arrhythmia, and heart failure, causing predominant deaths worldwide. Patients need a potential diagnostic system and treatment before cardiomyocyte damage. Cardiac biomarkers are released from myocytes immediately after a heart attack. Troponin is an efficient biomarker released from dead cells within a few hours. Aptamers are artificial antibodies used effectively in the biosensor field for biomarker detection. Along with aptamers, the application of nanomaterials is also expected to enhance the detection limits of biosensors. In this investigation, selected aptamers against cardiac troponin I (cTnI) were conjugated with gold nanoparticles (GNPs) to diagnose MI and compared with an aptamer-only control group on an interdigitated electrode surface. Based on electroanalysis, cTnI was detected at concentrations as low as 1 fM, and the detection limit improved to 100 aM when the aptamer was conjugated with GNP. In addition, aptamer-GNP conjugates increased the current level at the tested concentrations of cTnI. Control experiments with noncomplementary aptamers and relevant proteins did not result in notable changes in the current, demonstrating the selective detection of cTnI.

Heart Infection Prognosis Analysis by Two-dimensional Spot Tracking Imaging.

Cardiovascular death is one of the leading causes worldwide; an accurate identification followed by diagnosing the cardiovascular disease increases the chance of a better recovery. Among different demonstrated strategies, imaging on cardiac infections yields a visible result and highly reliable compared to other analytical methods. Two-dimensional spot tracking imaging is the emerging new technology that has been used to study the function and structure of the heart and test the deformation and movement of the myocardium. Particularly, it helps to capture the images of each segment in different directions of myocardial strain values, such as valves of radial strain, longitudinal strain, and circumferential strain. In this overview, we discussed the imaging of infections in the heart by using the two-dimensional spot tracking.

Aptamer-antibody dual probes on single-walled carbon nanotube bridged dielectrode: Comparative analysis on human blood clotting factor.

Haemophilia is a blood clotting disorder known as 'Christmas disease' caused when the blood has defect with the clotting factor(s). Bleeding leads various issues, such as chronic pain, arthritis and a serious complication during the surgery. Identifying this disease is mandatory to take the necessary treatment and maintains the normal clotting. It has been proved that the level of factor IX (FIX) is lesser with haemophilia patient and the attempt here is focused to quantify FIX level by interdigitated electrode (IDE) sensor. Single-walled carbon nanotube (SWCNT) was utilized to modify IDE sensing surface. On this surface, dual probing was evaluated with aptamer and antibody to bring the possible advantages. The detection limit with antibody was found to be 1 pM, while aptamer shows 100 fM. Further, a fine-tuning was attempted with sandwich pattern of aptamer-FIX-antibody and antibody-FIX-aptamer and compared. Specific elevation of detection with 10 folds was noticed and displayed the detection at 100 f. in both sandwich patterns. In addition, FIX was detected in the diluted human serum by aptamer-FIX-antibody sandwich, it was found that FIX detected from the dilution factor 1:640. A novel demonstration is with higher discrimination against other clotting factors, XI and VII.

Synthesis and characterization of reduced graphene oxide using the aqueous extract of Eclipta prostrata.

In this study, biological deoxygenation of graphene oxide (GO) using an Eclipta prostrata phytoextract was performed via the infusion method. The presence of oxide groups on the surface of graphene and removal of oxides groups by reduction were characterized through morphological and structural analyses. Field emission scanning electron microscopy images revealed that the synthesized GO and rGO were smooth and morphologically sound. Transmission electron microscopy images showed rGO developing lattice fringes with smooth edges and transparent sheets. Atomic force microscopy images showed an increase in the surface roughness of graphite oxide (14.29 nm) compared with that of graphite (1.784 nm) due to the presence of oxide groups after oxidation, and the restoration of surface roughness to 2.051 nm upon reduction. Energy dispersive X-ray analysis indicated a difference in the carbon/oxygen ratio between GO (1.90) and rGO (2.70). Fourier-transform infrared spectroscopy spectrum revealed peak stretches at 1029, 1388, 1578, and 1630 cm-1 for GO, and a decrease in the peak intensity after reduction that confirmed the removal of oxide groups. X-ray photoelectron microscopy also showed a decrease in the intensity of oxygen peak after reduction. In addition, thermogravimetric analysis suggested that rGO was less thermally stable than graphite, graphite oxide, and GO, with rGO decomposing after heating at temperatures ranging from room temperature to 600 °C.

MXene Surface on Multiple Junction Triangles for Determining Osteosarcoma Cancer Biomarker by Dielectrode Microgap Sensor.

BACKGROUND: In recent years, nanomaterials have justified their dissemination for biosensor application towards the sensitive and selective detections of clinical biomarkers at the lower levels. MXene is a two-dimensional layered transition metal, attractive for biosensing due to its chemical, physical and electrical properties along with the biocompatibility. MATERIALS AND METHODS: This work was focused on diagnosing osteosarcoma (OS), a common bone cancer, on MXene-modified multiple junction triangles by dielectrode sensing. Survivin protein gene is highly correlated with OS, identified on this sensing surface. Capture DNA was immobilized on MXene by using 3-glycidoxypropyltrimethoxysilane as an amine linker and duplexed by the target DNA sequence. RESULTS: The limitation and sensitivity of detection were found as 1 fM with the acceptable regression co-efficient value (y=1.0037⨰ + 0.525; R2=0.978) and the current enhancement was noted when increasing the target DNA concentrations. Moreover, the control sequences of single- and triple-mismatched and noncomplementary to the target DNA sequences failed to hybridize on the capture DNA, confirming the specificity. In addition, different batches were prepared with capture probe immobilized sensing surfaces and proved the efficient reproducibility. CONCLUSION: This microgap device with Mxene-modified multiple junction triangles dielectrode surface is beneficial to quantify the survivin gene at its lower level and diagnosing OS complication levels.

Aluminosilicate Nanocomposites from Incinerated Chinese Holy Joss Fly Ash: A Potential Nanocarrier for Drug Cargos.

An incredible amount of joss fly ash is produced from the burning of Chinese holy joss paper; thus, an excellent method of recycling joss fly ash waste to extract aluminosilicate nanocomposites is explored. The present research aims to introduce a novel method to recycle joss fly ash through a simple and straightforward experimental procedure involving acidic and alkaline treatments. The synthesized aluminosilicate nanocomposite was characterized to justify its structural and physiochemical characteristics. A morphological analysis was performed with field-emission transmission electron microscopy, and scanning electron microscopy revealed the size of the aluminosilicate nanocomposite to be ~25 nm, while also confirming a uniformly spherical-shaped nanostructure. The elemental composition was measured by energy dispersive spectroscopy and revealed the Si to Al ratio to be 13.24 to 7.96, showing the high purity of the extracted nanocomposite. The roughness and particle distribution were analyzed using atomic force microscopy and a zeta analysis. X-ray diffraction patterns showed a synthesis of faceted and cubic aluminosilicate crystals in the nanocomposites. The presence of silica and aluminum was further proven by X-ray photoelectron spectroscopy, and the functional groups were recognized through Fourier transform infrared spectroscopy. The thermal capacity of the nanocomposite was examined by a thermogravimetric analysis. In addition, the research suggested the promising application of aluminosilicate nanocomposites as drug carriers. The above was justified by an enzyme-linked apta-sorbent assay, which claimed that the limit of the aptasensing aluminosilicate-conjugated ampicillin was two-fold higher than that in the absence of the nanocomposite. The drug delivery property was further justified through an antibacterial analysis against Escherichia coli (gram-negative) and Bacillus subtilis (gram-positive).

Nanodetection of Head and Neck Cancer on Titanium Oxide Sensing Surface.

Head and neck cancer is a heterogeneous disease, originating in the squamous cells lining the larynx (voice box), mouth, pharynx (throat), nasal cavity and salivary glands. Head and neck cancer diagnosis at the later stage is greatly influencing the survival rate of the patient. It makes a mandatory situation to identify this cancer at the earlier stages of development with a suitable biomarker. Squamous cell carcinoma antigen (SCC-Ag) is a circulating serum tumour biomarker, and the elevated level has been found in the head and neck cancer patients and highly correlated with the tumour volume. The present research was carried out to detect and quantify the level of SCC-Ag on titanium oxide (TiO2)-modified interdigitated electrode sensor (IDE) by SCC-Ag antibody. The detection of SCC-Ag was found at the level of 100 fM, while it was improved to 10 fM when the antibody was conjugated with gold nanostar, representing a 10-fold improvement. Interestingly, this enhancement in sensitivity is 1000-folds higher than other substrates. Moreover, the specificity analysis was carried out using two different control proteins and noticed that the antibody only recognised SCC-Ag, indicating the specific detection on IDE-TiO2 sensing surface.

Detection of microRNA-335-5p on an Interdigitated Electrode Surface for Determination of the Severity of Abdominal Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) refers to the enlargement of the lower artery of the abdominal aorta, and identification of an early detection tool is urgently needed for diagnosis. In the current study, an interdigitated electrode (IDE) sensing surface was used to identify miRNA-335-5p, which reflects the formation of AAAs. The uniformity of the silica material was observed by 3D profilometry, and the chemically modified highly conductive surface improved the detection via the I-V mode. The targeted miRNA-335-5p was detected in a dose-dependent manner and based on linear regression and 3σ analyses, the sensitivity was determined to be 1 fM with a biotinylated probe. The high specificity was shown by discriminating the target sequence from noncomplementary and single- and triple-mismatched sequences. These outputs demonstrated the high-performance detection of miRNA-335-5p with good reproducibility for determination of the severity of AAA.