Building a Nucleic Acid Nanostructure with DNA-Epitope Conjugates for a Versatile Approach to Electrochemical Protein Detection.

The recent surge of effort in nucleic-acid-based electrochemical (EC) sensors has been fruitful, yet there remains a need for more generalizable EC platforms for sensing multiple classes of clinically relevant targets. We recently reported a nucleic acid nanostructure for simple, economical, and more generalizable EC readout of a range of analytes, including small molecules, peptides, proteins, and antibodies. The nanostructure is built through on-electrode enzymatic ligation of three oligonucleotides for attachment, binding, and signaling. However, the generalizable detection of larger proteins remains a challenge. Here, we adapted the sensor to quantify larger proteins in a more generic manner through conjugating the protein's minimized antibody-binding epitope to the central DNA strand. This concept was verified using creatine kinase (CK-MM), a biomarker of muscle damage and several disorders for which rapid clinical sensing is important. DNA-epitope conjugates permitted a competitive immunoassay for the CK protein at the electrode via square-wave voltammetry (SWV). Sensing through a signal-off mechanism, the anti-CK antibody limit of detection (LOD) was 5 nM with a response time as low as 3 min. Antibody displacement by native protein analytes gave a signal-on response with the CK sensing range from the LOD of 14 nM up to 100 nM, overlapping with the normal (nonelevated) human clinical range (3-37 nM), and the sensor was validated in 98% human serum. While a need for improved DNA-epitope conjugate purification was identified, overall, this approach allows the quantification of a generic protein- or peptide-binding antibody and should facilitate future quantitative EC readouts of clinically relevant proteins that were previously inaccessible to EC techniques.

Ionic Strength and Hybridization Position near Gold Electrodes Can Significantly Improve Kinetics in DNA-Based Electrochemical Sensors.

A variety of electrochemical (EC) biosensors play critical roles in disease diagnostics. More recently, DNA-based EC sensors have been established as promising for detecting a wide range of analyte classes. Since most of these sensors rely on the high specificity of DNA hybridization for analyte binding or structural control, it is crucial to understand the kinetics of hybridization at the electrode surface. In this work, we have used methylene blue-labeled DNA strands to monitor the kinetics of DNA hybridization at the electrode surface with square-wave voltammetry. By varying the position of the double-stranded DNA segment relative to the electrode surface as well as the bulk solution's ionic strength (0.125-1.00 M), we observed significant interferences with DNA hybridization closer to the surface, with more substantial interference at lower ionic strength. As a demonstration of the effect, toehold-mediated strand displacement reactions were slowed and diminished close to the surface, while strategic placement of the DNA binding site improved reaction rates and yields. This work manifests that both the salt concentration and DNA hybridization site relative to the electrode are important factors to consider when designing DNA-based EC sensors that measure hybridization directly at the electrode surface.

Electrochemical Sensing of the Peptide Drug Exendin-4 Using a Versatile Nucleic Acid Nanostructure.

Although endogenous peptides and peptide-based therapeutics are both highly relevant to human health, there are few approaches for sensitive biosensing of this class of molecules with minimized workflow. In this work, we have further expanded on the generalizability of our recently developed DNA nanostructure architecture by applying it to electrochemical (EC) peptide quantification. While DNA-small molecule conjugates were used in a prior work to make sensors for small molecule and protein analytes, here DNA-peptide conjugates were incorporated into the nanostructure at the electrode surfaces, and antibody displacement permitted rapid peptide sensing. Interestingly, multivalent DNA-peptide conjugates were found to be detrimental to the assay readout, yet these effects could be minimized by solution-phase bioconjugation. The final biosensor was validated for quantifying exendin-4 (4.2 kDa)─a human glucagon-like peptide-1 receptor agonist important in diabetes therapy─for the first time using EC methods with minimal workflow. The sensor was functional in 98% human serum, and the low nanomolar assay range lies between the injected dose concentration and the therapeutic range, boding well for future applications in therapeutic drug monitoring.

A Comparative Study Between Septal Quilting Sutures Without Nasal Packing and Only Nasal Packing Post-septal Correction.

Septal correction is the commonest surgery by rhinologists worldwide. We aimed at studying the comfort level of the patient with standard postoperative nasal packing with Merocel and placing quilting sutures in septum leaving the nose unpacked in the postoperative period. We conducted the study in the tertiary care centre enrolling 82 patients in a quasi-randomised method of odd and even numbers placing them in the nasal packing group and the quilting group respectively. We used analogue scoring method for subjective assessment of comfort level in the postoperative period and the surgeon objectively assessed the patient on follow up. The results were tabulated and analysed. Postoperative pain, headache and sleep disturbance was significantly more in the nasal packing group. We found that the crusting is commonly seen in patients in the nasal packing group. Quilting the nasal septum and leaving the nasal cavity unpacked increases the comfort level of the patient in the postoperative period. The resultant pain, headache and sleep disturbance caused by nasal packing can be significantly avoided by using quilting the septum without nasal packing. We also observed that by avoiding nasal packing postoperatively, the patients were more comfortable and compliant with the treatment regimen and follow-up.

Nonfaradaic Current Suppression in DNA-Based Electrochemical Assays with a Differential Potentiostat.

One of the key factors limiting sensitivity in many electrochemical assays is the nonfaradaic or capacitive current. This is particularly true in modern assay systems based on DNA monolayers at gold electrode surfaces, which have shown great promise for bioanalysis in complex milieu such as whole blood or serum. While various changes in analytical parameters, redox reporter molecules, DNA structures, probe coverage, and electrode surface area have been shown useful, background reduction by hardware subtraction has not yet been explored for these assays. Here, we introduce new electrochemistry hardware that considerably suppresses nonfaradaic currents through real-time analog subtraction during current-to-voltage conversion in the potentiostat. This differential potentiostat (DiffStat) configuration is shown to suppress or remove capacitance currents in chronoamperometry, cyclic voltammetry, and square-wave voltammetry measurements applied to nucleic acid hybridization assays at the electrode surface. The DiffStat makes larger electrodes and higher sensitivity settings accessible to the user, providing order-of-magnitude improvements in sensitivity, and it also significantly simplifies data processing to extract faradaic currents in square-wave voltammetry (SWV). Because two working electrodes are used for differential measurements, unique arrangements are introduced such as converting signal-OFF assays to signal-ON assays or background drift correction in 50% human serum. Overall, this new potentiostat design should be helpful not only in improving the sensitivity of most electrochemical assays, but it should also better support adaptation of assays to the point-of-care by circumventing complex data processing.

A Nucleic Acid Nanostructure Built through On-Electrode Ligation for Electrochemical Detection of a Broad Range of Analytes.

For an assay to be most effective in point-of-care clinical analysis, it needs to be economical, simple, generalizable, and free from tedious workflows. While electrochemistry-based DNA sensors reduce instrumental costs and eliminate complicated procedures, there remains a need to address probe costs and generalizability, as numerous probes with multiple conjugations are needed to quantify a wide range of biomarkers. In this work, we have opened a route to circumvent complicated multiconjugation schemes using enzyme-catalyzed probe construction directly on the surface of the electrode. With this, we have created a versatile DNA nanostructure probe and validated its effectiveness by quantification of proteins (streptavidin, anti-digoxigenin, anti-tacrolimus) and small molecules (biotin, digoxigenin, tacrolimus) using the same platform. Tacrolimus, a widely prescribed immunosuppressant drug for organ transplant patients, was directly quantified with electrochemistry for the first time, with the assay range matching the therapeutic index range. Finally, the stability and sensitivity of the probe was confirmed in a background of minimally diluted human serum.

Understanding Signal and Background in a Thermally Resolved, Single-Branched DNA Assay Using Square Wave Voltammetry.

Electrochemical bioanalytical sensors with oligonucleotide transducer molecules have been recently extended for quantifying a wide range of biomolecules, from small drugs to large proteins. Short DNA or RNA strands have gained attention recently due to the existence of circulating oligonucleotides in human blood, yet challenges remain for adequately sensing these targets at electrode surfaces. In this work, we have developed a quantitative electrochemical method which uses target-induced proximity of a single-branched DNA structure to drive hybridization at an electrode surface, with readout by square-wave voltammetry (SWV). Using custom instrumentation, we first show that precise control of temperature can provide both electrochemical signal amplification and background signal depreciation in SWV readout of small oligonucleotides. Next, we thoroughly compared 25 different combinations of binding energies by their signal-to-background ratios and differences. These data served as a guide to select the optimal parameters of binding energy, SWV frequency, and assay temperature. Finally, the influence of experimental workflow on the sensitivity and limit of detection (LOD) of the sensor is demonstrated. This study highlights the importance of precisely controlling temperature and SWV frequency in DNA-driven assays on electrode surfaces while also presenting a novel instrumental design for fine-tuning of such systems.

A reusable electrochemical proximity assay for highly selective, real-time protein quantitation in biological matrices.

Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)-as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation.

Bell's palsy in Singapore: a view from the patient's perspective

<p><b>INTRODUCTION</b>Bell's palsy is a well-recognised disease with robust research on its possible aetiologies and epidemiology, but scant information on patients' concerns and concepts regarding the condition is available. We aimed to evaluate the ideas, concerns and expectations of patients with Bell's palsy in Singapore.</p><p><b>METHODS</b>A cross-sectional study was conducted at a single tertiary-care hospital in Singapore. Participants were all patients with newly diagnosed Bell's palsy referred to the otolaryngology department either from the emergency department or by general practitioners. Participants were given a self-administered questionnaire and their facial nerve palsies were graded by the consultant doctor.</p><p><b>RESULTS</b>A total of 52 patients were recruited, of which 41 were available for analysis. 78.0% of patients were concerned that they were having a stroke upon presentation of the symptoms. Other beliefs about the cause of the disease included overwork or stress (36.6%), something that the patient had eaten (9.8%) and supernatural forces (2.4%). About 50% of patients had tried some form of complementary or alternative therapy other than the steroids/medicines prescribed by their general practitioner or emergency physician. While 39.0% of patients agreed that the Internet had helped them understand more about their condition in addition to the information provided by the physician, 9.8% of them specifically disagreed with this statement.</p><p><b>CONCLUSION</b>We have found that patients with Bell's palsy in Singapore are not very knowledgeable about the disease. Although the Internet is a useful resource, a physician's explanation of the disease and its natural progression remains of utmost importance.</p>