Malonyl-CoA serves as the main building block for the biosynthesis of many important polyketides, as well as fatty acid-derived compounds, such as biofuel. Escherichia coli, Corynebacterium gultamicum, and Saccharomyces cerevisiae have recently been engineered for the biosynthesis of such compounds. However, the developed processes and strains often have insufficient productivity. In the current study, we used enzyme-engineering approach to improve the binding of acetyl-CoA with ACC. We generated different mutations, and the impact was calculated, which reported that three mutations, that is, S343A, T347W, and S350W, significantly improve the substrate binding. Molecular docking investigation revealed an altered binding network compared to the wild type. In mutants, additional interactions stabilize the binding of the inner tail of acetyl-CoA. Using molecular simulation, the stability, compactness, hydrogen bonding, and protein motions were estimated, revealing different dynamic properties owned by the mutants only but not by the wild type. The findings were further validated by using the binding-free energy (BFE) method, which revealed these mutations as favorable substitutions. The total BFE was reported to be -52.66 ± 0.11 kcal/mol for the wild type, -55.87 ± 0.16 kcal/mol for the S343A mutant, -60.52 ± 0.25 kcal/mol for T347W mutant, and -59.64 ± 0.25 kcal/mol for the S350W mutant. This shows that the binding of the substrate is increased due to the induced mutations and strongly corroborates with the docking results. In sum, this study provides information regarding the essential hotspot residues for the substrate binding and can be used for application in industrial processes.
Abdominal aortic aneurysm (AAA), a medical complication, occurs when the aortic area becomes swollen and very large. It is mandatory to identify AAA to avoid the breakdown of aneurysms. C-reactive protein (CRP) has been recognized as one of the biomarkers for identifying AAA due to the possibility of CRP produced in vascular tissue, which contributes to the formation of an aneurysm, and it is elevated in patients with a ruptured AAA. This research work was designed to develop an immunosensor on a multiwalled carbon nanotube (MWCNT)-modified surface to quantify the CRP level. Anti-CRP specificity was constructed on the MWCNT surface through a silane linker to interact with CRP. The detection limit of CRP was calculated as 100 pM with an R2 (determination coefficient) value of 0.9855 (y = 2.3446x - 1.9922) on a linear regression graph. The dose-dependent linear pattern was registered from 200 to 3000 pM and attained the saturation level during binding at 3000 pM. Furthermore, serum-spiked CRP showed a clear increase in the current response, proving the specific recognition of CRP in biological samples. This designed biosensor identifies CRP at a lower level and can help diagnose AAA.
C-reactive protein (CRP) is an acute phase reactant to be a marker of inflammation and has been correlated with the cardiac injury. An immunoassay was performed using anti-human CRP antibody on an InterDigitated electrode (IDE) sensor to determine and specify CRP concentration for diagnosing the condition of myocardial inflammation. To promote the detection, gold nanoparticle (GNP) was seeded on the aminated-IDE surface. Anti-CRP was hitched on the GNP-seeded surface and identified the abundance of CRP. The limit of quantification was found as 100 fM, and the higher current response was noticed by increasing CRP concentrations with the sensitivity at 1 pM. Furthermore, CRP-spiked human serum did not interfere the determination of CRP and increased the current response, indicating suitability for a real-life sample. Similarly, the control experiments with nonimmune antibody Troponin I are not showing the definite current responses, proving the selective identification of CRP. This method of diagnosing is needful to determine the cardiovascular injury at the right time.
The E6 region has higher protuberant probability annealing than consensus probe focusing on another region in the human papillomavirus (HPV) genome in terms of detection and screening method. Here, we designed the first multiple virus single-stranded deoxyribonucleic acid (ssDNA) for multiple detections in an early phase of screening for cervical cancer in the E6 region and became a fundamental evolution of detection electrochemical HPV biosensor. Gene profiling of the virus ssDNA sequences has been carried by high-end bioinformatics tools such as GenBank, Basic Local Alignment Searching Tools (BLAST), and Clustal OMEGA in a row. The output from bioinformatics tools resulted in 100% of similarities between our virus ssDNA probe and HPV complete genome in the databases. The cross-validation between HPV genome and our designed virus ssDNA provided high specificity and selectivity during screening methods compared with Pap smear. The DNA probe for HPV 18, 5' COOH-GAT CCA GAA GGT ACA GAC GGG GAG GGC ACG 3', while 5'COOH-GGG CGC TGT GCA GTG TGT TGG AGA CCC CGA3' as DNA probe for HPV 58 designed with 66.77% guanine (G) and cytosine (C) content for both. Our virus ssDNA probe for the HPV biosensor promises high sensitivity, specificity, selectivity, repeatability, low fluid consumption, and will be useful in mini-size diagnostic devices for cervical cancer detection.
Osteoporosis, a bone disease is caused by the deterioration of bone and shows an enhanced risk of bone fracture and decreasing bone mineral density. Unfortunately, the available radiological techniques are expensive, and have disadvantages such as radiation intake, need a specialist to handle the instrument, and so forth. This research is focused to develop a point-of-care system to identify osteocalcin on current-volt sensor, which helps to diagnose the bone metabolism and prognostics. Antiosteocalcin antibody was attached on the electrode through the silane-modified iron material. The antibody-immobilized sensing surface was utilized to identify the level of osteocalcin and the detection limit of 100 pg/ml reached on linear concentrations of 0.01-3000 ng/ml. Calculations were made by triplicates (n = 3; 3δ) on the determination coefficient of y = 0.2637x-0.6012; R2 = 0.9319. Further, control proteins failed to bind with immobilized antibody, confirmed by the specific osteocalcin detection. This research is to identify the osteoporosis biomarker and to help determine the conditions with osteoporosis.
Current developments in sensors and actuators are heralding a new era to facilitate things to happen effortlessly and efficiently with proper communication. On the other hand, Internet of Things (IoT) has been boomed up with er potential and occupies a wide range of disciplines. This study has choreographed to design of an algorithm and a smart data-processing scheme to implement the obtained data from the sensing system to transmit to the receivers. Technically, it is called "telediagnosis" and "remote digital monitoring," a revolution in the field of medicine and artificial intelligence. For the proof of concept, an algorithmic approach has been implemented for telediagnosis with one of the degenerative diseases, that is, Parkinson's disease. Using the data acquired from an improved interdigitated electrode, sensing surface was evaluated with the attained sensitivity of 100 fM (n = 3), and the limit of detection was calculated with the linear regression value coefficient. By the designed algorithm and data processing with the assistance of IoT, further validation was performed and attested the coordination. This proven concept can be ideally used with all sensing strategies for immediate telemedicine by end-to-end communications.
The urinary C-terminal telopeptide fragment of type II collagen (uCTX-II) has been reported as the efficient blood-based biomarker for osteoarthritis, which affects knees, hands, spine, and hips. This study reports a sensing strategy with antibody-conjugated gold nanoparticles (GNP) on an interdigitated electrode (IDE) to determine uCTX-II. The GNP-antibody complex was chemically immobilized on the IDE surface through the amine linker. uCTX-II was determined by monitoring the alteration in current upon interacting the GNP-complexed antibody. This strategy was improved the detection by attracting higher uCTX-II molecules, and the detection limit falls in the range of 10-100 pM with an acceptable regression value [y = 0.6254x - 0.4073, R² = 0.9787]. The sensitivity of the detection was recognized at 10 pM. Additionally, upon increasing the uCTX-II concentration, the current changes were increased in a linear fashion. Control detection with nonimmune antibody and control protein do not increase the current level, confirming the specific detection of uCTX-II. This method of detection helps in diagnosing osteoarthritis and its follow-up treatment.
Mycoplasma pneumoniae is a highly infectious bacterium and the major cause of pneumonia especially in school-going children. Mycoplasma pneumoniae affects the respiratory tract, and 25% of patients experience health-related problems. It is important to have a suitable method to detect M. pneumoniae, and gold nanoparticle (GNP)-based colorimetric biosensing was used in this study to identify the specific target DNA for M. pneumoniae. The color of GNPs changes due to negatively charged GNPs in the presence of positively charged monovalent (Na+ ) ions from NaCl. This condition is reversed in the presence of a single-stranded oligonucleotide, as it attracts GNPs but not in the presence of double-stranded DNA. Single standard capture DNA was mixed with optimal target DNA that cannot be adsorbed by GNPs; under this condition, GNPs are not stabilized and aggregate at high ionic strength (from 100 mM). Without capture DNA, the GNPs that were stabilized by capture DNA (from 1 μM) became more stable under high ionic conditions and retaining their red color. The GNPs turned blue in the presence of target DNA at concentrations of 1 pM, and the GNPs retained a red color when there was no target in the solution. This method is useful for the simple, easy, and accurate identification of M. pneumoniae target DNA at higher discrimination and without involving sophisticated equipment, and this method provides a diagnostic for M. pneumoniae.
One of the current issues with thyroid tumor is early diagnosis as it makes the higher possibility of curing. This research was focused to detect and quantify the level of specific target sequence complementation of miR-222 with capture DNA sequence on interdigitated electrode (IDE) sensor. The aluminum electrode with the gap and finger sizes of 10 µm was fabricated on silicon wafer, further the surface was amine-functionalized for accommodating carboxylated-DNA probe. With DNA-target RNA complementation, the detection limit was attained to be 1 fM as estimated by a linear regression analysis [y = 1.5325x - 2.1171 R² = 0.9065] and the sensitivity was at the similar level. Current responses were higher by increasing the target RNA sequence concentrations. Control experiments with mismatched/noncomplementary sequences were failed to complement the capture DNA sequence immobilized on IDE, indicating the specific target validation. This research helps diagnosing and identifying the progression with thyroid tumor and miRNA being a potential "marker" in atypia diagnosis.
Osteosarcoma is a bone cancer formed by the cells of the bone. Children, young adults, and teens are highly affected by osteosarcoma. Early identification of osteosarcoma is mandatory to improve the treatment and increase the lifespan of the patients. MicroRNA-195 (miR-195) was shown to be a suitable biomarker for osteosarcoma, and the present study describes a sensitive method of miR-195 identification by nuclease digestion in ELISA to detect and quantify the level of miR-195. S1 nuclease catalyzed endo- and exonucleolytic digestion of single-stranded (ss) RNA and DNA on ELISA polystyrene substrate, which helped to identify duplexed miR-195. This method selectively and specifically identified miR-195 without any biofouling interactions and reached the limit of detection at 10 fM within the range from 10 fM to 10 nM. Due to complete digestion of ssDNA, single- and triple-mismatched sequences failed to increase the ELISA signal, indicating specific miRNA detection. Furthermore, human serum spiked with miR-195 did not interfere with the detection, confirming selective identification. This method identified miR-195 at a lower level and will help to diagnose earlier stages of osteosarcoma.
A highly sensitive silica-alumina (Si-Al)-modified capacitive non-Faradaic glucose biosensor was introduced to monitor gestational diabetes. Glucose oxidase (GOx) was attached to the Si-Al electrode surface as the probe through amine-modification followed by glutaraldehyde premixed GOx as aldehyde-amine chemistry. This Si-Al (∼50 nm) modified electrode surface has increased the current flow upon binding of GOx with glucose. Capacitance values were increased by increasing the glucose concentrations. A mean capacitance value was plotted and the detection limit was found as 0.03 mg/mL with the regression coefficient value, R² = 0.9782 [y = 0.8391x + 1.338] on the linear range between 0.03 and 1 mg/mL. Further, a biofouling experiment with fructose and galactose did not increase the capacitance, indicating the specific glucose detection. This Si-Al-modified capacitance sensor detects a lower level of glucose presence and helps in monitoring gestational diabetes.
17β-Estradiol-E2 (17β-E2) is a steroid hormone that plays a major role in the reproductive endocrine system and is involved in various processes, such as pregnancy, fertility, and menopause. In this study, the performance of an enzyme-linked immunosorbent assay (ELISA) for 17β-E2 quantification was enhanced by using a gold nanoparticle (GNP)-conjugated aptamer. An anti-17β-E2-aptamer-GNP antibody was immobilized on an amine-modified ELISA surface. Then, 17β-E2 was allowed to interact with and be sandwiched by antibodies. Aptamer-GNP conjugation was confirmed by colorimetric assays via the naked eye and UV-visible light spectroscopy. The detection limit based on a signal-to-noise ratio (S/N) of 3 was estimated to be 1.5 nM (400 pg/mL), and the linear range was 1.5-50 nM. Control experiments (without 17β-E2/with a complementary aptamer sequence/with a nonimmune antibody) confirmed the specific detection of 17β-E2. Moreover, 17β-E2 spiking of human serum did not interrupt the interaction between 17β-E2 and its antibody and aptamer. Thus, the developed ELISA can be used as an alternate assay for quantification of 17β-E2 and assessment of endocrine-related gynecological problems.
Rheumatoid arthritis (RA) is an autoimmune disorder causing chronic inflammation in the small joints of the articular bone and destruction of articular cartilage. RA causes stiffness, pain, joint destruction, substantial comorbidity, and functional disability. Early-stage diagnosis of RA can help in the treatment of the disease and expand the patient life span. Interleukins are a group of inflammatory cytokines; in particular, an abundance of interleukin-6 (IL-6) was found in the synovial fluid and serum. In RA patients, the levels of IL-6 have been found to be correlated with the disease, and this work focused on detecting IL-6 by its aptamer with the help of a biotin-streptavidin strategy on an interdigitated electrode. A sensitivity of 1 fM (0.021 pg/mL) and a limit of detection of 10 fM (0.21 pg/mL) were found by a linear regression [y = 0.6413x - 0.6249; R² = 0.952] of the linear range from 1 fM to 100 pM. This method enhanced the immobilization of higher aptamer molecules for recognizing RA in serum-containing samples and is applicable to other diseases.
Thyroid cancer appears in endocrine glands and specific to thyroid glands has been reported widely. This work was targeted to identify and quantify thyroglobulin by using antithyroglobulin antibody complexed silane surface on interdigitated electrode (IDE) sensing surface. (3-Aminopropyl)triethoxysilane linker was used to make silane-coupling with antibody and attached on the hydroxylated IDE. This electroanalytical IDE revealed the dose-dependent responses with thyroglobulin concentrations. By getting increments with the thyroglobulin concentrations, the current responses were enhanced concomitantly and the thyroglobulin detection limit was noted as 1 pM on the linear curve [y = 0.1311x + 0.5386; R² = 0.9707] with the sensitivity at lower picomolar range. Moreover, the control experiments with thyroid peroxidase and nonimmune antibody cannot yield any response of current, confirming the specific detection of thyroglobulin. This research set-up is useful to determine and quantify the thyroglobulin and diagnose thyroid cancer.
Acute myocardial infarction (AMI) is the heart attack happening when the blood flow is terminated to the heart muscles. C-reactive protein (CRP) level is raising significantly in AMI patients after the onset of symptom; also, temporal variations of CRP in plasma of AMI patient have also been found. Quantifying the concentration of CRP helps to identify the condition associated with AMI. Plasmonic enzyme-linked immunosorbent assay (ELISA) was utilized here to identify CRP by the sandwich of aptamer and antibody. Bare-eye CRP detection was achieved by plasmonic ELISA through the aggregation (blue color) of gold nanoparticle in the presence of CRP, whereas in the absence of CRP, it retains its red color (dispersion). Depending on the catalase presence on the ELISA surface, hydrogen peroxide (H2 O2 ) controls gold growth and differentiates with color changes. To achieve the lowest detection limit of CRP, H2 O2 (200 µM), gold seed (0.2 µM), and streptavidin-catalase (1:500) were found optimal. The detection limit was reached at 0.25 µg/mL, whereas it was 0.5 µg/mL in the CRP-spiked serum. This method of detection system is easier to detect the levels of CRP and helps diagnosing AMI.
Abdominal aortic aneurysm (AAA) is a serious, life-threatening vascular disease that presents as an enlarged area of the aorta, which is the main artery that carries blood away from the heart. AAA may occur at any location in the aorta, but it is mainly found in the abdominal region. A ruptured AAA causes serious health issues, including death. Traditional imaging techniques, such as computed tomography angiogram, magnetic resonance imaging, and ultrasound sonography, have been used to identify AAAs. Circulating biomarkers have recently become attractive for diagnosing AAAs due to their cost-effectiveness compared to imaging. Insulin-like growth factor 1 (IGF-1), a secreted hormone vital for human atherosclerotic plaque stability, has been found to be an efficient biomarker for AAA identification. In this report, immunosensing was performed by using an InterDigitated electrode (IDE) sensor to detect circulating levels of IGF-1. The detection limit of IGF-1 was found to be 100 fM with this sensor. Moreover, related protein controls (IGF-2 and IGFBP3) were not detected with the same antibody, indicating selective IGF-1 detection. Thus, immunosensing by using an IDE sensor may help to effectively diagnose AAAs and represents a basic platform for further development.
In biomedical implant technology, nanosurface such as titania nanotube arrays (TNA) could provide better cellular adaptation, especially for long-term tissue acceptance response. Mechanotransduction activities of TNA nanosurface could involve the cytoskeleton remodeling mechanism. However, there is no clear insight into TNA mechano-cytoskeleton remodeling activities, especially computational approaches. Epithelial cells have played critical interface between biomedical implant surface and tissue acceptance, particularly for long-term interaction. Therefore, this study investigates genomic responses that are responsible for cell-TNA mechano-stimulus using epithelial cells model. Findings suggested that cell-TNA interaction may improve structural and extracellular matrix (ECM) support on the cells as an adaptive response toward the nanosurface topography. More specifically, the surface topography of the TNA might improve the cell polarity and adhesion properties via the interaction of the plasma membrane and intracellular matrix responses. TNA nanosurface might engross the cytoskeleton remodeling activities for multidirectional cell movement and cellular protrusions on TNA nanosurface. These observations are supported by the molecular docking profiles that determine proteins' in silico binding mechanism on TNA. This active cell-surface revamping would allow cells to adapt to develop a protective barrier toward TNA nanosurface, thus enhancing biocompatibility properties distinctly for long-term interaction. The findings from this study will be beneficial toward nano-molecular knowledge of designing functional nanosurface technology for advanced medical implant applications.
Myocardial infarction (MI) is highly related to cardiac arrest leading to death and organ damage. Radiological techniques and electrocardiography have been used as preliminary tests to diagnose MI; however, these techniques are not sensitive enough for early-stage detection. A blood biomarker-based diagnosis is an immediate solution, and due to the high correlation of troponin with MI, it has been considered to be a gold-standard biomarker. In the present research, the cardiac biomarker troponin I (cTnI) was detected on an interdigitated electrode sensor with various surface interfaces. To detect cTnI, a capture aptamer-conjugated gold nanoparticle probe and detection antibody probe were utilized and compared through an alternating sandwich pattern. The surface metal oxide morphology of the developed sensor was proven by microscopic assessments. The limit of detection with the aptamer-gold-cTnI-antibody sandwich pattern was 100 aM, while it was 1 fM with antibody-gold-cTnI-aptamer, representing 10-fold differences. Further, the high performance of the sensor was confirmed by selective cTnI determination in serum, exhibiting superior nonfouling. These methods of determination provide options for generating novel assays for diagnosing MI.
A "Janus particle" refers to the production of two materials in a single system and shows a difference in physical characteristics, and two surfaces participate in the formation with different chemistries. This research generated the Janus using a hybrid of zinc oxide (ZnO) and gold (Au) on the sensor surface toward making high-performance DNA sensors. The Janus ZnO/Au-textured film was synthesized via the one-step sol-gel method, which involves a suitable ratio of a mixture of ZnO sol seed solution. The synthesized Janus ZnO/Au-textured film undergoes a low-temperature aqueous hydrothermal route to synthesize quasi-one-dimensional nanowires. The average grain size in the Janus ZnO/Au nanotextured wire was 41.60 nm. The fabricated nanotextured wire was further optimized by tuning the thickness and characterized by XRD and high-resolution microscopy. Electrical characterization was conducted on the Janus ZnO/Au nanotextured wire coupled with an interdigitated electrode sensor to detect the specific leptospirosis DNA strand. The generated device is capable of detecting lower DNA concentration at 1 × 10-13 M with a sensitivity of 8.54 MΩ M-1 cm-2 . The high performance is attained on linear concentrations of 10-6 -10-13 M with the determination coefficient, "I = 135437.63C-3609.07" R2 = 0.9551. A potential strategy is proposed as a base for developing different high-performance sensors.
Lymphatic filariasis is a neglected parasitic disease that affects millions in tropical and subtropical countries and is caused by Wuchereria and Brugia species. Specific and sensitive detection methods are essential in mapping infected areas where rapid tests are needed to cover underdeveloped and remote regions, which facilitates eliminating the disease as a public health problem. A few commercialized rapid tests based on antigen or antibody detection are available, but the former only detects infection by Wuchereria species and cross-reacts with nonlymphatic filaria, whereas antibody detection might provide positive results of previous infection. Here, we report the production of three different recombinant immunoglobulin gamma (IgG)1 antibodies based on scFvs previously generated via human antibody phage display technology, that is, anti-BmR1 clone 4, anti-BmXSP clone 5B, and anti-BmXSP clone 2H2. The scFv sequences were cloned into a pCMV-IgG1 vector, then transfected into a HEK293F cell line. The generated antibodies were found to be able to bind to their respective targets even at relatively low concentration. Conjugation of Fc to scFv induces binder stability and provides multiple labeling sites for probes and signaling molecules that can be used in rapid tests.