Portable contactless caliper.

This work presents a portable optical meter for noncontact thickness measurement. The device shines a focused laser light on a thin and transparent sample, resulting in an interference between light reflecting from the top and from the bottom surface, and the interfering pattern is recorded by a linear sensor array before data analysis with an Arduino microcontroller. The device produced accurate thickness values from glass cover slips and transparent plastic sheets within a fraction of a second per measurement. Additionally, the sample's refractive index is not required a priori. Therefore, it has a high potential to be of use in real-time quality control in transparent thick-film coating and manufacturing.

Glucose Oxidase/Egg White Protein Microparticles with a Redox Mediator for Glucose Biosensors on a Screen-Printed Electrode and a Decomposable Electrode.

Glucose oxidase (GOx) is a typical model enzyme used to create biosensors. Exploring a strategy to prepare ready-to-use functional enzymatic microparticles combining GOx and food-based proteins offers compelling advantages. However, no reports exist on the integration of egg white materials to synthesize functional biorecognition particles with glucose oxidation catalytic functions for electrochemical biosensors. Here, we demonstrate functional microparticles combining egg white proteins, GOx, and 9,10-phenanthrenequinone (PQ). The egg white proteins crosslink to form three-dimensional scaffolds to accommodate GOx and redox molecules. The PQ mediator enhances electron transfer between the electrode surface and the GOx enzyme's flavin adenine dinucleotides. The functional microparticles are directly applied to the printed electrode. The performance of these microparticles is evaluated using a screen-printed carbon nanotube (CNT)-modified electrode coated with GOx/PQ/egg white protein microparticles. The analytical performance of the system exhibits a linear range of 0.125-40 mM, with a maximum current (Imax) and a Michaelis-Menten constant (Km) being 0.2 µA and 4.6 mM, respectively. Additionally, a decomposable electrode composed of CNTs and edible oil conjugated with functional enzyme microparticles is shown to undergo degradation under gastric conditions. Utilizing food-based proteins to accommodate enzymes and to create redox-active microparticles for catalyzing glucose oxidation offers advantages in developing affordable and degradable bioelectrodes. This concept holds promise for advancing biocompatible electrodes in biosensor and bioelectronics applications.

The Development of the Fluorescence-based Portable Device for Lead (II) and Formalin Determination in Food Samples by Using Nitrogen-Doped Carbon Dots (N-CDs).

The fluorescence-based portable device for the determination of lead (Pb2+) and formalin (FA) in food samples by using Nitrogen-doped carbon dots (N-CDs) as a fluorescence probe was developed. The proposed approach, Pb2+, and FA were determined based on the photo-induced electron transfer (PET) mechanism and the silver mirror reaction. The fluorescence intensity of the N-CDs decreased with the increase of Pb2+ concentration and increased with the increasing FA concentration. The fluorescence intensity of N-CDs after the reactions were measured by a filter-free fluorometer platform using a commercial camera module and a Raspberry Pi, a compact computer, as a detector and processor. The experimental results were obtained using control samples with known Pb2+ and FA concentrations in the 0.01-10 mg L- 1 and 25-150 mg L- 1, respectively. The proposed approach is simple, low-cost, and accurate for the on-site monitoring of Pb2+ and FA in various food samples. Of utmost importance, the proposed approach is expected to be a pioneering model for the future development of other analytes with a broad range of practical applications.

Portable device for dual detection of fluorescence and absorbance for biosensing or chemical sensing applications.

Early detection is key to prevent health problems and could be performed by biosensors and chemical sensors. However, a lot of them still need bulky benchtop equipment. This work presents a portable device for measuring fluorescence and light absorption that can be used with optical biosensors or chemical sensors. It uses a small laser diode as a light source and three filter-mounted photodiodes as detectors, all of which are inexpensive, customizable and widely available commercially. The results from our device show good correlation with that from commercial instruments. Therefore, it could be beneficial for early or on-site detection.

Fluorescent cysteine probe based on a signal amplification unit, a catalyzed hairpin assembly reaction and Förster resonance energy transfer.

This work developed a sensitive DNA-based fluorescent probe comprising a cysteine binding unit and a signal amplification unit based on a catalyzed hairpin assembly (CHA) reaction. The cysteine binding unit comprises a homodimer of single-stranded DNA (ssDNA) rich in cytosine and held together by silver ions. In the presence of cysteine, the homodimer is disintegrated because of cysteine-silver binding that liberates the ssDNA, which drives the CHA reaction in the signal amplification unit. Förster resonance energy transfer (FRET) was used to report the generation of the amplified double-stranded DNA (dsDNA) product. Under the optimal conditions, the probe provided a good linearity (100-1200 nM), a good detection limit (47.8 ± 2.7 nM) and quantification limit (159.3 ± 5.3 nM), and a good sensitivity (1.900 ± 0.045µM-1). The probe was then used to detect cysteine in nine real food supplement samples. All results provided good recoveries that are acceptable by the AOAC, indicating that it has potential for practical applications.

Newly found K+-Thioflavin T competitive binding to DNA G-quadruplexes and the development of a label-free fluorescent biosensor with extra low detection limit for K+ determination in urine samples.

The determination of potassium ion K+ in body fluids is important in health monitoring and diagnoses. One of the interesting and simple methods for K+ detection is the use of label-free biosensors based on DNA G-quadruplexes (GQs) coupled with a specific fluorescent probe, such as Thioflavin T (ThT), which lights up when bound with K+-stabilized GQs. However, these biosensors are not generally sensitive. In this work, we found a solution: at a low concentration, K+ competes with ThT in binding to a bimolecular GQ or a tetramolecular GQ, resulting in a decrease in ThT fluorescence emission with increasing K+. Therefore, we developed a label-free turn-off fluorescent K+ sensor. The sensor provides a very low detection limit of 21.87 ± 0.59 nM. Other possible interfering components in urine did not exert any effect even at quantities that were 10-fold greater than their upper limit of normal concentrations found in urine samples. With its only requirement of diluting samples, the developed low-cost label-free probe and simple sensor was successfully applied to the direct detection of K+ in normal urine samples with high accuracy (recoveries ranged from 90% to 100%).

Smartphone-based portable fluorescence sensor with gold nanoparticle mediation for selective detection of nitrite ions.

A simple, portable device for the detection of NO2- via a fluorescence method was developed. The proposed device consisted of a dark box containing a blue LED as a low-power excitation light source and a smartphone with a mobile application for RGB analysis as a light detector. Detection was mediated by using synthesized cetyltrimethylammonium bromide-stabilized gold nanoparticles (CTAB-AuNPs). The CTAB-AuNPs were etched with NO2- to yield Au3+, which catalyzes the oxidation of o-phenylenediamine (OPD) in the presence of H2O2 to generate 2,3-diaminophenazine (DAP). Triton X-100 (TX-100) micelles were introduced to improve the DAP fluorescence emission. The fluorescence intensity of DAP was recorded by the smartphone in terms of RGB intensity, which was correlated with the NO2- concentration. This method provided a wide linear working concentration range (0.5-100 µM), a limit of detection of 0.17 µM and excellent selectivity for NO2- over other anions.

A copper nanoclusters probe for dual detection of microalbumin and creatinine.

A fluorescent probe based on glutathione-capped copper nanoclusters (GSH-CuNCs) was developed for the detection of dual targets, human serum albumin (HSA) and creatinine, in human urine. The GSH-CuNCs were synthesized by a one-pot green method using ascorbic acid as a reducing agent. The detection of HSA was in a turn-on mode via electrostatic interaction in a basic condition while the detection of creatinine was in a turn-off mode via non-covalent bonding in an acidic condition. Under optimal conditions, the linear range and detection limit of HSA were 5.0 nM to 150 nM and 1.510 ±â€¯0.041 nM, while those of creatinine were 30 µM to 1000 µM and 13.0 ±â€¯1.0 µM. This easily fabricated nanocluster probe provided a fast response with high sensitivity, and good selectivity. Recoveries from urine samples were in the range of 81.44 ±â€¯0.25 to 109.22 ±â€¯0.57% for HSA and 80.57 ±â€¯0.16 to 109.0 ±â€¯0.10% for creatinine. The urinary analytical results from the fluorescent probe were in good agreement (P > 0.05) to those obtained from immunoturbidimetric and enzymatic methods, signifying the excellent performance of this sensing system.

Real-time investigation of the roles of ATP hydrolysis by UvrA and UvrB during DNA damage recognition in nucleotide excision repair.

Nucleotide excision repair (NER) stands out among other DNA repair systems for its ability to process a diverse set of unrelated DNA lesions. In bacteria, NER damage detection is orchestrated by the UvrA and UvrB proteins, which form the UvrA2-UvrB2 (UvrAB) damage sensing complex. The highly versatile damage recognition is accomplished in two ATP-dependent steps. In the first step, the UvrAB complex samples the DNA in search of lesion. Subsequently, the presence of DNA damage is verified within the UvrB-DNA complex after UvrA has dissociated. Although the mechanism of bacterial NER damage detection has been extensively investigated, the role of ATP binding and hydrolysis by UvrA and UvrB during this process remains incompletely understood. Here, we report a pre-steady state kinetics Förster resonance energy transfer (FRET) study of the real-time interaction between UvrA, UvrB, and damaged DNA during lesion detection. By using UvrA and UvrB mutants harboring site-specific mutations in the ATP binding sites, we show for the first time that the dissociation of UvrA from the UvrAB-DNA complex does not require ATP hydrolysis by UvrB. We find that ATP hydrolysis by UvrA is not essential, but somehow facilitates the formation of UvrB-DNA complex, with ATP hydrolysis at the proximal site of UvrA playing a more critical role. Consistent with previous reports, our results indicated that the ATPase activity of UvrB is essential for the formation of UvrB-DNA complex but is not required for the binding of the UvrAB complex to DNA.

Fluorescence quenching by photoinduced electron transfer between 7-methoxycoumarin and guanine base facilitated by hydrogen bonds: an in silico study.

In this study, the effects of hydrogen bond (H-bond) formation on fluorescence quenching of 7-methoxycoumarin (7MC) via photo-induced electron transfer from a guanine base (Gua) are investigated using a combined quantum mechanics/molecular mechanics simulation. The electronic structure is calculated by the floating occupation molecular orbital complete active space configuration interaction modification on a semiempirical method. Then the full multiple spawning method is employed for the dynamics simulations on multiple electronic states. The methods employed here are validated by simulating direct dynamics of 7MC (without Gua) and compared with available experimental results. Our computational results are in good agreement with the previously reported experimental results in terms of spectroscopic properties of 7MC. In the case of a H-bonded 7MC-Gua complex, the results from constrained dynamics simulations and single-point calculations suggest that the electron transfer occurs on the second excited state and it depends not only on the H-bond length but also on the intermolecular planarity between 7MC and Gua. Moreover, a proton coupled electron transfer can occur at ≈1 Å of H-bond length, where a proton from Gua is also transferred together with the electron to 7MC. The obtained simulations are expected to be greatly beneficial for designing effective fluorescently labeled nucleotide probes as well as providing information for precise fluorescence signal interpretation.

Wavelet analysis on time-frequency plane of optical coherence tomography: simultaneous signal quality improvement in structural and velocity images.

In this Letter, we utilize one-dimensional wavelet analysis to improve the quality of morphology images and velocity profiles of optical coherence tomography simultaneously, by performing analysis on the complex time-frequency plane of raw interferograms, prior to image construction. The results indicate a robust signal improvement that also preserves accuracy for both morphology and velocity information and has been demonstrated on a variety of samples with diverse flow speeds and morphologies.

A FRET based aptasensor coupled with non-enzymatic signal amplification for mercury (II) ion detection.

In this work, the idea of incorporating a non-enzymatic signal amplification with a regular aptasensor was tested. In this proof of principle, the sensor was designed for the detection of mercury (II) ions (Hg(2+)) based on the Förster Resonance Energy Transfer (FRET), and the catalyzed hairpin assembly (CHA) technique that was used as the signal amplification method. This sensor comprised a mercury aptamer-catalyst complex (Apt-C) and two types of hairpin DNA: H1 labeled with fluorescein and H2 labeled with tetramethylrhodamine. In the presence of Hg(2+), two facing thymine bases in the mercury aptamer strand were coordinated with one mercury ion. This caused the release of the catalyst for the catalyzed hairpin assembly (CHA) reaction that turned H1 and H2 hairpins into H1-H2 hybrids. FRET was then used to report the hairpin-duplex transformation. The sensor showed excellent specificity towards Hg(2+) over other possible interfering cations present at even a 100 fold greater concentrations. It had a linear range of 10.0-200.0nM, and a good detection limit of 7.03±0.18nM, which is lower than the regulatory mercury limit for drinking water (10nM or 2ppb). The sensor was used to detect spiked Hg(2+) in nine real surface water samples collected from three different areas. Acceptable recoveries and small standard deviations indicated that the sensor was practically applicable, and the proposed idea to incorporate a CHA amplification in a regular aptasensor was not only feasible but beneficial. The same principles can be applied to develop sensors for various different targets.

Enhancing capacitive DNA biosensor performance by target overhang with application on screening test of HLA-B*58:01 and HLA-B*57:01 genes.

A highly sensitive label-free DNA biosensor based on PNA probes immobilized on a gold electrode was used to detect a hybridization event. The effect of a target DNA overhang on the hybridization efficiency was shown to enhance the detected signal and allowed detection at a very low concentration. The sensors performances were investigated with a complementary target that had the same length as the probe, and the signal was compared to the target DNAs with different lengths and overhangs. A longer target DNA overhang was found to provide a better response. When the overhang was on the electrode side the signal enhancement was greater than when the overhang was on the solution side due to the increased thickness of the sensing surface, hence produced a larger capacitance change. Using conformationally constrained acpcPNA probes, double stranded DNA was detected sensitively and specifically without any denaturing step. When two acpcPNA probes were applied for the screening test for the double stranded HLA-B*58:01 and HLA-B*57:01 genes that are highly similar, the method differentiated the two genes in all samples. Both purified and unpurified PCR products gave comparable results. This method would be potentially useful as a rapid screening test without the need for purification and denaturation of the PCR products.

A new screening method for flunitrazepam in vodka and tequila by fluorescence spectroscopy.

A new screening method for flunitrazepam in colourless alcoholic beverages based on a spectroscopic technique is proposed. Absorption and steady-state fluorescence of flunitrazepam and its protonated form with various acids were investigated. The redshift of the wavelength of maximum absorption was distinctively observed in protonated flunitrazepam. An emissive fluorescence at 472 nm was detected in colourless spirits (vodka and tequila) at room temperature. 2-M perchloric acid was the most appropriated proton source. By using electron ionization mass spectrometry and time-dependent density functional theory calculations, the possible structure of protonated flunitrazepam was identified to be 2-nitro-N-methylacridone, an acridone derivative as opposed to 2-methylamino-5-nitro-2'-fluorobenzophenone, a benzophenone derivative.

A novel reconfigurable optical biosensor based on DNA aptamers and a DNA molecular beacon.

In order to alter a typical molecular aptamer beacon (MAB) to detect a different analyte there is currently a need to change the whole sensor unit including the expensive labeling fluorophores. In this work a DNA-based reconfigurable molecular aptamer beacon was developed. It is composed of two parts: a variable part and a constant part. The variable part comprises an aptamer strand and its complementary strand while the constant part is an oligonucleotide doubly labeled with a Förster Resonance Energy Transfer (FRET) pair and the two parts become joined via DNA hybridization. The sensor exists in two conformations: a folded (high FRET) and an unfolded (low FRET) in the absence and presence of the aptamer-target binding respectively. This sensor can be reconfigured by washing away the aptamer and the complementary strand using proper complementary strands, called washers. As a proof of the principle, a sensor that bound the enzyme thrombin, an analyte with a strong binding, was first constructed and then reconfigured to bind adenosine, selected as an analyte with a weak binding. We believe that the design is of universal use applicable to many types of aptamers.

Advances in single-molecule fluorescence methods for molecular biology.

Ever since their introduction two decades ago, single-molecule (SM) fluorescence methods have matured and branched out to address numerous biological questions, which were inaccessible via ensemble measurements. Among the current arsenal, SM fluorescence techniques have capabilities of probing the dynamic interactions of nucleic acids and proteins via Förster (fluorescence) resonance energy transfer (FRET), tracking single particles over microns of distances, and deciphering the rotational motion of multisubunit systems. In this exciting era of transitioning from in vitro to in vivo and in situ conditions, it is anticipated that SM fluorescence methodology will become a common tool of molecular biology.

Rapid frequency-domain FLIM spinning disk confocal microscope: lifetime resolution, image improvement and wavelet analysis.

A spinning disk confocal attachment is added to a full-field real-time frequency-domain fluorescence lifetime-resolved imaging microscope (FLIM). This provides confocal 3-D imaging while retaining all the characteristics of the normal 2-D FLIM. The spinning disk arrangement allows us to retain the speed of the normal 2-D full field FLIM while gaining true 3-D resolution. We also introduce the use of wavelet image transformations into the FLIM analysis. Wavelets prove useful for selecting objects according to their morphology, denoising and background subtraction. The performance of the instrument and the analysis routines are tested with quantitative physical samples and examples are presented with complex biological samples.

Single molecule nanometronome.

We constructed a DNA-based nanomechanical device called the nanometronome. Our device is made by introducing complementary single-stranded overhangs at the two arms of the DNA four-way junction. The ticking rates of this stochastic metronome depend on ion concentrations and can be changed by a set of DNA-based switches to deactivate/reactivate the sticky end. Since the device displays clearly distinguishable responses even with a single base pair difference, it may lead to a single molecule sensor of minute sequence differences of a target DNA.