Customizable Nichrome Wire Heaters for Molecular Diagnostic Applications.

Accurate sample heating is vital for nucleic acid extraction and amplification, requiring a sophisticated thermal cycling process in nucleic acid detection. Traditional molecular detection systems with heating capability are bulky, expensive, and primarily designed for lab settings. Consequently, their use is limited where lab systems are unavailable. This study introduces a technique for performing the heating process required in molecular diagnostics applicable for point-of-care testing (POCT), by presenting a method for crafting customized heaters using freely patterned nichrome (NiCr) wire. This technique, fabricating heaters by arranging protrusions on a carbon black-polydimethylsiloxane (PDMS) cast and patterning NiCr wire, utilizes cost-effective materials and is not constrained by shape, thereby enabling customized fabrication in both two-dimensional (2D) and three-dimensional (3D). To illustrate its versatility and practicality, a 2D heater with three temperature zones was developed for a portable device capable of automatic thermocycling for polymerase chain reaction (PCR) to detect Escherichia coli (E. coli) O157:H7 pathogen DNA. Furthermore, the detection of the same pathogen was demonstrated using a customized 3D heater surrounding a microtube for loop-mediated isothermal amplification (LAMP). Successful DNA amplification using the proposed heater suggests that the heating technique introduced in this study can be effectively applied to POCT.

Highly sensitive plasmonic paper substrate fabricated via amphiphilic polymer self-assembly in microdroplet for detection of emerging pharmaceutical pollutants.

We report an innovative and facile approach to fabricating an ultrasensitive plasmonic paper substrate for surface-enhanced Raman spectroscopy (SERS). The approach exploits the self-assembling capability of poly(styrene-b-2-vinyl pyridine) block copolymers to form a thin film at the air-liquid interface within the single microdroplet scale for the first time and the subsequent in situ growth of silver nanoparticles (AgNPs). The concentration of the block copolymer was found to play an essential role in stabilizing the droplets during the mass transfer phase and formation of silver nanoparticles, thus influencing the SERS signals. SEM analysis of the morphology of the plasmonic paper substrates revealed the formation of spherical AgNPs evenly distributed across the surface of the formed copolymer film with a size distribution of 47.5 nm. The resultant enhancement factor was calculated to be 1.2 × 107, and the detection limit of rhodamine 6G was as low as 48.9 pM. The nanohybridized plasmonic paper was successfully applied to detect two emerging pollutants-sildenafil and flibanserin-with LODs as low as 1.48 nM and 3.45 nM, respectively. Thus, this study offers new prospects for designing an affordable and readily available, yet highly sensitive, paper-based SERS substrate with the potential for development as a lab-on-a-chip device.

Emerging Trends of Bilirubin Oxidases at the Bioelectrochemical Interface: Paving the Way for Self-Powered Electrochemical Devices and Biosensors.

Bilirubin oxidases (BODs) [EC 1.3.3.5 - bilirubin: oxygen oxido-reductase] are enzymes that belong to the multicopper oxidase family and can oxidize bilirubin, diphenols, and aryl amines and reduce the oxygen by direct four-electron transfer from the electrode with almost no electrochemical overpotential. Therefore, BOD is a promising bioelectrocatalyst for (self-powered) biosensors and/or enzymatic fuel cells. The advantages of electrochemically active BOD enzymes include selective biosensing, biocatalysis for efficient energy conversion, and electrosynthesis. Owing to the rise in publications and patents, as well as the expanding interest in BODs for a range of physiological conditions, this Review analyzes scientific literature reports on BOD enzymes and current hypotheses on their bioelectrocatalysis. This Review evaluates the specific research outcomes of the BOD in enzyme (protein) engineering, immobilization strategies, and challenges along with their bioelectrochemical properties, limitations, and applications in the fields of (i) biosensors, (ii) self-powered biosensors, and (iii) biofuel cells for powering bioelectronics.

Screening of antioxidant capacity of Nepali medicinal plants with a novel singlet oxygen scavenging assay.

Pollutant exposure due to industrial development increases oxidative stress in human bodies. Dietary intake of antioxidant shows a protective effect against oxidative damage induced by oxidative stress. Therefore, the development of natural antioxidants is needed. In this study, the antioxidant activities of some Nepali medicinal plant extracts were measured. Using Rose bengal and 3,3',5,5'-tetramethylbenzidine, a novel assay was utilized to evaluate the singlet oxygen scavenging capacity, and showed a strong correlation with other antioxidant assays. Also, antioxidant capacities based on four assays including the singlet oxygen scavenging assay were highly correlated (≥ 0.858) with the total phenolic contents in the medicinal plant extracts. Among the selected extracts, Persicaria capitata, Elaphoglossum marginatum and Eurya acuminata showed the highest antioxidant capacities. Overall, this study presents a novel approach for evaluating singlet oxygen scavenging capacity, and performed a screening of antioxidant capacities of 54 Nepali herbal medicines. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01175-z.

Effect of gelation technique on lipid digestibility of emulsion-loaded alginate microparticles: a systematic review and meta-analysis.

Alginate microparticles fabricated via calcium gelation or layer-by-layer assembly are commonly used for encapsulating emulsions. In this study, the impact of these two gelation methods on the lipid digestibility of emulsions was reviewed through a systematic screening of relevant studies. From the literature search (Scopus, PubMed, and Web of Science databases), 604 records were screened and 25 articles were included in the analysis. The fold change of free fatty acid release rate at the end of in vitro digestion process between alginate-encapsulated emulsion and emulsions not encapsulated by alginate was calculated for calcium gelation (weighted mean of response ratio 0.64, 95% CI 0.54-0.75) and layer-by-layer assembly (weighted mean of response ratio 0.89, 95% CI 0.81-0.98). Alginate-calcium hydrogels showed stronger inhibition of the extent of lipid digestion than alginate-coated multilayer emulsions. The structural and particle size differences between alginate microparticles acquired using different techniques may contribute to this phenomenon.

Effects of encapsulation methods on bioaccessibility of anthocyanins: a systematic review and meta-analysis.

Anthocyanins have multiple health benefits. However, they are prone to degradation during gastrointestinal digestion, impeding their utilization. Various encapsulation systems have been proposed to improve their bioaccessibility and bioavailability. This review aims to provide a systematic evaluation and meta-analysis of published studies examining the effect of microencapsulation on the bioaccessibility of anthocyanins. A comprehensive and systematic literature search of three databases (Scopus, PubMed, and Web of Science) was conducted. Studies were selected according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses criteria and were reviewed independently by two investigators. Overall, 34 articles were included in the systematic review and 24 were included in the meta-analysis. The fold changes in bioaccessibility between encapsulated and non-encapsulated anthocyanins from eligible studies were calculated. The median and 95% confidence intervals (CI) of the fold changes for spray-drying (median 1.23, 95% CI 0.91-1.92), freeze-drying (median 1.19, 95% CI 0.61-1.28), simple coacervation (median 1.80, 95% CI 1.41-3.20), and complex coacervation (median 1.61, 95% CI 0.21-25.00) were calculated. Simple coacervation showed a promising protection against degradation during in vitro digestion. However, when a large number of anthocyanins cannot be released from the microparticles during digestion, encapsulation impedes the bioaccessibility of anthocyanins.

Pressed Lateral Flow Assay Strips for Flow Delay-Induced Signal Enhancement in Lateral Flow Assay Strips.

This paper proposes that the signal intensity of a lateral flow assay (LFA) strip can be increased by pressing the top of the strip, effectively reducing its flow rate. The reduced flow rate allows more time for antigen-antibody interactions to occur, resulting in increased signal intensity and an improved detection limit. To assess the potential of the pressed LFA (pLFA) strip, C-reactive protein (CRP) diluted in phosphate-buffered saline (PBS) and serum is detected, affording signal enhancement and a lowered limit of detection. Additionally, to show that the signal enhancement by pressure-induced flow delay applies to existing LFA products, commercially available COVID-19 antigen test strips are pressed, and signal enhancement is observed. Lastly, we show that the signal intensity of COVID-19 LFA kits can be increased by approximately two-fold at maximum by applying pressure on top of the manufactured product. This study suggests that pressed LFA strips can be used to reduce the chances of determining ambiguous signals as false-negative results and can potentially improve the detection sensitivity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-022-00085-w.

A carbon-black-embedded poly(dimethylsiloxane)-paper hybrid device for energy-efficient nucleic-acid amplification in point-of-care testing.

A paper-based device patterned with a carbon-black-poly(dimethylsiloxane) (PDMS) mixture is developed as a heating platform for nucleic-acid amplification tests. The photothermal effect of carbon black under 808 nm laser irradiation is used to conduct loop-mediated isothermal amplification (LAMP) to detect Escherichia coli (E. coli) O157:H7, a foodborne pathogen. We characterize the heat generation of carbon black by changing its concentration and the hardness of PDMS. Then, we optimize the minimum laser power required to perform LAMP. The proposed paper-based device requires less than 15 min to perform LAMP, and the result can be confirmed based on the color change observed by the naked eye. The rfbE gene of E. coli O157:H7 is specifically amplified, with a detection limit of 102 CFU mL-1. Amplification is also performed by using a laboratory-made laser-diode device, which consumes only 2 W h during its operation. The low cost, disposability, and easy fabrication of the paper-based device make it a powerful tool for point-of-care testing.

Real-time monitoring of mono- and dual-species biofilm formation and eradication using microfluidic platform.

In a human host, bacterial Staphylococcus aureus and fungal Candida albicans pathogens form a mixed biofilm that causes severe mortality and morbidity. However, research on the formation and eradication of mixed biofilms under dynamic conditions is lacking. Thus, this study employed a microfluidic technique to analyze the real-time formation of mono- and dual-species (S. aureus and C. albicans) biofilms and noninvasive optical treatment of the established mature biofilm using 405-nm laser light. A herringbone mixer thoroughly mixed both bacterial and fungal cells in the growth media before being injected into the observation channels on the microfluidic chip. At a flow rate of 1.0 µL/min of growth media for 24 h, the bacterial biofilm coverage was up to 15% higher than that of the fungal biofilm (50% for bacteria vs. 35% for fungus). On the other hand, the dual-species biofilm yielded the highest coverage of ~ 96.5% because of the collective interaction between S. aureus and C. albicans. The number of cell proliferation events in S. aureus was higher than that of C. albicans for 12 h, which indicates that the S. aureus biofilm was developed faster than C. albicans. The novel in situ test platform showed a significant bactericidal effect (80%) of the 405-nm laser light at 1080 J/cm2 towards the established S. aureus biofilm, whereas the same treatment removed approximately 69% of the mixed cells in the dual-species biofilm. This study revealed that the developed microfluidic platform could be utilized to monitor the formation of dual-species biofilms in real-time and laser-induced antimicrobial effects on dual-species biofilms.

Simultaneous Measurement of pH and Temperature with Phase-Shifted Long-Period Fiber Grating Inscribed on High-Birefringence Fiber by CO2 Laser Pulses.

We propose an optical fiber grating sensor capable of simultaneously measuring pH and temperature based on a phase-shifted long-period fiber grating (PS-LPFG) inscribed on high-birefringence fiber (HBF). The PS-LPFG was fabricated on HBF with CO2 laser pulses, and a phase shift π was induced by inserting a grating-free fiber region (GFFR) between two identical LPFGs with a grating period of ˜510 µm. The length of the GFFR was set as half of the grating period to induce a π phase shift. With the spectral characteristics of a π-PS-LPFG exhibiting two split attenuation bands, the PS-LPFG written on HBF, which is referred to as the HB-PS-LPFG, can create two polarization-dependent transmission spectra with dual-resonance dips at different wavelengths according to two orthogonal input polarization states, e.g., linear horizontal polarization (LHP) and linear vertical polarization (LVP). For simultaneous measurement of pH and temperature with the fabricated HB-PS-LPFG as a sensor head, the inter-resonance wavelength separation of the dual-resonance dips in each transmission spectrum obtained for an LHP or LVP input signal was exploited as a sensor indicator. By investigating the wavelength changes of the two sensor indicators, which were induced by pH and temperature variations, linear and independent spectral responses to both pH and temperature variations were experimentally confirmed in a pH range from 1 to 11 and a temperature range from 25 to 65 °C. Owing to the unique pH and temperature responses of the fabricated HB-PS-LPFG, ambient variations in pH and temperature could be simultaneously estimated from the measured wavelength changes and sensitivities of the two sensor indicators.

Development and validation of new poisoning mortality score system for patients with acute poisoning at the emergency department.

BACKGROUND: A prediction model of mortality for patients with acute poisoning has to consider both poisoning-related characteristics and patients' physiological conditions; moreover, it must be applicable to patients of all ages. This study aimed to develop a scoring system for predicting in-hospital mortality of patients with acute poisoning at the emergency department (ED). METHODS: This was a retrospective analysis of the Injury Surveillance Cohort generated by the Korea Center for Disease Control and Prevention (KCDC) during 2011-2018. We developed the new-Poisoning Mortality Scoring system (new-PMS) to generate a prediction model using the derivation group (2011-2017 KCDC cohort). Points were computed for categories of each variable. The sum of these points was the new-PMS. The validation group (2018 KCDC cohort) was subjected to external temporal validation. The performance of new-PMS in predicting mortality was evaluated using area under the receiver operating characteristic curve (AUROC) for both the groups. RESULTS: Of 57,326 poisoning cases, 42,568 were selected. Of these, 34,352 (80.7%) and 8216 (19.3%) were enrolled in the derivation and validation groups, respectively. The new-PMS was the sum of the points for each category of 10 predictors. The possible range of the new-PMS was 0-137 points. Hosmer-Lemeshow goodness-of-fit test showed adequate calibration for the new-PMS with p values of 0.093 and 0.768 in the derivation and validation groups, respectively. AUROCs of the new-PMS were 0.941 (95% CI 0.934-0.949, p < 0.001) and 0.946 (95% CI 0.929-0.964, p < 0.001) in the derivation and validation groups, respectively. The sensitivity, specificity, and accuracy of the new-PMS (cutoff value: 49 points) were 86.4%, 87.2%, and 87.2% and 85.9%, 89.5%, and 89.4% in the derivation and validation groups, respectively. CONCLUSIONS: We developed a new-PMS system based on demographic, poisoning-related variables, and vital signs observed among patients at the ED. The new-PMS showed good performance for predicting in-hospital mortality in both the derivation and validation groups. The probability of death increased according to the increase in the new-PMS. The new-PMS accurately predicted the probability of death for patients with acute poisoning. This could contribute to clinical decision making for patients with acute poisoning at the ED.

Low-cost, open-source 3D printed antibody dispenser for development and small-scale production of lateral flow assay strips.

We present a low cost, 3D printed open-source antibody dispenser that can be easily built and used for the development of lateral flow assay (LFA) strips. The fabrication of LFA strips need dispensing of antibodies or antigens in a linear fashion and commercially available dispensers typically cost from few thousands to few tens of thousands of US dollars. In this paper, an antibody dispenser was built by using 3D printed and commercially available parts, which cost no more than 30 USD. This paper presents a detailed instruction on how to assemble the printer and how to achieve a specific line width for the dispensed antibody. By using syringe needles of different gauges, line width ranging from 0.23 to 1.8 mm can be dispensed, and by varying the speed controller, the dispensing needle's speed can be varied between 2.8 and 3.8 cm/s. We demonstrate uniform dispensing of anti-C-reactive protein (CRP) antibody and anti-rabbit antibody to draw a test line and a control line, which are used for the detection of CRP. The dispenser can also be equipped with two dispensing needles to allow simultaneous dispensing of multiple reagents, which can be useful for LFA strip development.

A homobifunctional imidoester-based microfluidic system for simultaneous DNA and protein isolation from solid or liquid biopsy samples.

The isolation of bio-molecules such as proteins and nucleic acids is a necessary step for both diagnostic and analytical processes in the broad fields of research and clinical applications. Although a myriad of isolation technologies have been developed, a method for simultaneous protein and nucleic acid isolation has not been explored for clinical use. Obtaining samples from certain cancers or rare diseases can be difficult. In addition, the heterogeneity of cancer tissues typically leads to inconsistent results when analyzing biomolecules. We here describe a homobifunctional imidoester (HI)-based microfluidic system for simultaneous DNA and protein isolation from either a solid or liquid single biopsy sample. An efficient and cost effective microfluidic design with less air bubbles was identified among several candidates using simulation and experimental results from the streamlining of isolation processing. HI groups were used as capture reagents for the simultaneous isolation of bio-molecules from a single specimen in a single microfluidic system. The clinical utility of this system for the simultaneous isolation of DNA and proteins within 40 min was validated in cancer cell lines and 23 tissue biopsies from colorectal cancer patients. The quantity of isolated protein and DNA was high using this system compared to the spin-column method. This HI-based microfluidic system shows good rapidity, affordability, and portability in the isolation of bio-molecules from limited samples for subsequent clinical analysis.

Multiplexed Detection of Foodborne Pathogens from Contaminated Lettuces Using a Handheld Multistep Lateral Flow Assay Device.

This paper presents a handheld device that is capable of simplifying multistep assays to perform sensitive detection of foodborne pathogens. The device is capable of multiplexed detection of Escherichia coli (E. coli) O157:H7, Salmonella Typhimurium (S. Typhimurium), Staphylococcus aureus, and Bacillus cereus. The limit of detection for each bacterium was characterized, and then, the detection of bacteria from contaminated fresh lettuces was demonstrated for two representative foodborne pathogens. We employed a sample pretreatment protocol to recover and concentrate target bacteria from contaminated lettuces, which can detect 1.87 × 104 CFU of E. coli O157:H7 and 1.47 × 104 CFU of S. Typhimurium/1 g of lettuce without an enrichment process. Lastly, we demonstrated that the limit of detection can be reduced to 1 CFU of E. coli O157:H7 and 1 CFU of S. Typhimurium/1 g of lettuce by including a 6 h enrichment of contaminated lettuces in growth media before pretreatment.

Lateral flow assay-based bacterial detection using engineered cell wall binding domains of a phage endolysin.

The development of a cost-effective and efficient bacterial detection assay is essential for diagnostic fields, particularly in resource-poor settings. Although antibodies have been widely used for bacterial capture, the production of soluble antibodies is still expensive and time-consuming. Here, we developed a nitrocellulose-based lateral flow assay using cell wall binding domains (CBDs) from phage as a recognition element and colloidal gold nanoparticles as a colorimetric signal for the detection of a model pathogenic bacterium, Bacillus cereus (B. cereus). To improve conjugation efficiency and detection sensitivity, cysteine-glutathione-S-transferase-tagged CBDs and maltose-binding protein-tagged CBDs were produced in Escherichia coli (E. coli) and incorporated in our assays. The sensitivity of the strip to detect B. cereus was 1×104 CFU/mL and the overall assay time was 20min. The assay showed superior results compared to the antibody-based approach, and did not show any significant cross-reactivity. This proof of concept study indicates that the lateral flow assay using engineered CBDs hold considerable promise as simple, rapid, and cost-effective biosensors for whole cell detection.

Functional Packaging of Lateral Flow Strip Allows Simple Delivery of Multiple Reagents for Multistep Assays.

This paper presents a rotary device designed for facile delivery of multiple reagents to a paper strip for multistep assays. Its purpose is to allow users to easily perform multistep assays and achieve sensitive detection. While the test strip remains stationary, rotating the top piece of the device aligns the reagent and absorbent pads to each end of the paper strip and initiates fluid flow. Further incremental rotation makes an adjacent pair of pads to align simultaneously, causing fluid flow of subsequent reagent that was preloaded in the reagent pad. In this work, various porous substrates were tested to observe their effect on overall flow rate of the system and multistep assays were performed to demonstrate its simple use. As a proof of concept, enzyme-linked immunosorbent assay was carried out to detect Escherichia coli O157:H7.

Pressed Paper-Based Dipstick for Detection of Foodborne Pathogens with Multistep Reactions.

This paper presents a pressed paper-based dipstick that enables detection of foodborne pathogens with multistep reactions by exploiting the delayed fluid flow and channel partition formation on nitrocellulose (NC) membrane. Fluid behaviors are easily modified by controlling the amount of pressure and the position of pressed region on the NC membrane. Detection region of the dipstick is optimized by controlling flow rate and delayed time based on Darcy's law. All the reagents required for assay are dried on the NC membrane and they are sequentially rehydrated at the prepartitioned regions when the device is dipped into sample solution. In this manner, multistep reactions can be facilitated by one-step dipping of the dipstick into the sample solution. As a proof of concept, we performed detection of two fatal foodborne pathogens (e.g., Escherichia coli O157:H7 and Salmonella typhimurium) with signal enhancement. In addition, we expanded the utilization of channel partitions by developing a pressed paper-based dipstick into dual detection format.

Experimental Analysis of Porosity and Permeability in Pressed Paper.

In this paper, we report an analysis of pressed paper in terms of porosity and permeability. Previously, we reported a pressed paper that exhibits decreased porosity and permeability. Additionally, its applications into programmed sample delivery as well as flow rate control were reported. However, there is a need for a theoretical analysis of pressed paper in terms of porosity and permeability for a more precise design principle and its applications because porosity and permeability are important factors in determining fluidic behavior. Here, we propose a theoretical model for analyzing decreased porosity and permeability in pressed paper. Porosity and permeability of pressed paper were quantitatively calculated using experimental results with a theoretical model. Furthermore, based on the analyzed results of porosity and permeability in pressed paper, a porosity⁻permeability relationship was investigated.

Engineering the shape and structure of materials by fractal cut.

In this paper we discuss the transformation of a sheet of material into a wide range of desired shapes and patterns by introducing a set of simple cuts in a multilevel hierarchy with different motifs. Each choice of hierarchical cut motif and cut level allows the material to expand into a unique structure with a unique set of properties. We can reverse-engineer the desired expanded geometries to find the requisite cut pattern to produce it without changing the physical properties of the initial material. The concept was experimentally realized and applied to create an electrode that expands to >800% the original area with only very minor stretching of the underlying material. The generality of our approach greatly expands the design space for materials so that they can be tuned for diverse applications.

Programmed sample delivery on a pressurized paper.

This paper reports a method to control the fluid flow in paper-based microfluidic devices simply by pressing over the channel surface of paper, thereby decreasing the pore size and permeability of a non-woven polypropylene sheet. As a result, fluid resistance is increased in the pressed region and causes flow rate to decrease. We characterize the decrease of flow rate with respect to different amounts of pressure applied, and up to 740% decrease in flow velocity was achieved. In addition, we demonstrate flow rate control in a Y-shaped merging paper and sequential delivery of multiple color dyes in a three-branched paper. Furthermore, sequential delivery of multiple fluid samples is performed to demonstrate its application in multi-step colorimetric immunoassay, which shows a 4.3-fold signal increase via enhancement step.