Bioelectrochemical detection of L-lactate respiration using genetically modified Hansenula polymorpha yeast cells overexpressing flavocytochrome b2

In general, L-lactate respiration is difficult to detect in living yeast cells due to the small activity of L-lactate oxidizing enzymes within the mitochondria. Genetically modified cells of methylotrophic yeast Hansenula polymorpha overproducing L-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, also known as flavocytochrome b₂, FC b₂) were physically immobilized by means of a dialysis membrane onto various types of electrode materials in order to investigate the possibility of electrochemically detecting L-lactate respiration. It could be shown that in the case of genetically modified Hansenula polymorpha cells in contrast to cells from the parental strain, enhanced L-lactate-dependent respiration could be detected. Due to overproduction of FC b₂ the O₂ reduction current is decreased upon addition of L-lactate to the electrolyte solution. The electron transfer pathway in the L-lactate-dependent respiration process involves a cascade over three redox proteins, FC b₂, cytochrome c and Complex-IV, starting with L-lactate oxidation and ending with oxygen reduction. By means of selective inhibition of Complex IV with CN⁻, lactate respiration could be proven for causing the decrease in the O₂ reduction.     

Continuous Lactate Measurement that Combines a Portable Ultrafiltration Storage Device with an Enzyme Sensor

ABSTRACT|A simple portable continuous L-lactate monitoring system combining elegant ultrafiltration sample collection and storage with a lactate biosensor has been developed.|The sensor has a detection range from 0.05mM to 30mM lactate dependent on different assignments of the PCS hydrogel complex layer.|The sensor shows excellent performance on sensitivity and validity as well as operational stability over one month.|Samples containing different concentrations of lactate were collected in six hours with an ultrafiltration probe and stored in a 6m long PEEK tube of 125μm in internal diameter.|The ultraslow flow rate of 100 to 350nl/min was controlled by adjusting the restriction in the portable plastic syringe pump and a constant flow can be maintained over 24 hours.|The collected samples were then detected after reversing the flow with a lactate sensor.

The total weight of the collection system is only 8g, allowing free movement of the person being tested.     

Binding Characteristics Study of DNA based Aptamers for E. coli O157:H7

E. coli O157:H7 is a pathogenic bacterium producing verotoxins that could lead to serious complications such as hemolytic uremia syndrome. Fast detection of such pathogens is important. For rapid detection, aptamers are quickly gaining traction as alternative biorecognition molecules besides conventional antibodies. Several DNA aptamers have been selected for E. coli O157:H7. Nonetheless, there has not been a comparative study of the binding characteristics of these aptamers. In this work, we present a comprehensive analysis of binding characteristics including binding affinity (K_d) and binding capacity (B_max) of DNA-based aptamers for E. coli O157:H7 using qPCR. Our results show that aptamer E18R has the highest binding capacity to E. coli 157:H7 and the highest specificity over non-pathogenic E. coli strains K12 and DH5α. Our study also finds that the common biotin-tag modification at 5′ end typically changes the binding capacity significantly. For most of the selected aptamers, the binding capacity after a biotin-tag modification decreases. There exists a discrepancy in the binding capability between the selected aptamer and the aptamer used for detection. Our study also shows that a lower concentration of Mg²⁺ ions in the binding buffer leads to a decrease in the binding capacity of E17F and E18R, while it does not affect the binding capacity of S1 and EcoR1.     

Reagentless lactate electrodes based on electrocatalytic oxidation of flavocytochrome b2

Flavocytochrome b₂ (L-lactate :cytochrome c reductase, E.C. 1.1.2.3) from Hansenula anomala was entrapped on the surface of electrodes modified with various kinds of carbon black. The electrocatalytic oxidation of a reduced enzyme by the electroactive surface groups of carbon black enables this enzyme electrode to be used for the determination of lactate. The electrodes operate at ?0.2 to ?0.1 V vs. SCE (pH 7.0), which is low enough to avoid interference from ascorbic acid. Linear calibration graphs up to 0.5 mM lactate were obtained. Electrochemical measurements of lactate in human blood plasma and cell culture fluids showed good agreement with the results of spectrophotometric measurements.     

Aptamer-based label-free method for hemin recognition and DNA assay by capillary electrophoresis with chemiluminescence detection

An aptamer-based label-free approach to hemin recognition and DNA assay using capillary electrophoresis with chemiluminescence detection is introduced here. Two guanine-rich DNA aptamers were used as the recognition element and target DNA, respectively. In the presence of potassium ions, the two aptamers folded into the G-quartet structures, binding hemin with high specificity and affinity. Based on the G-quartet–hemin interactions, the ligand molecule was specifically recognized with a K _d ≈ 73 nM, and the target DNA could be detected at 0.1 μM. In phosphate buffer of pH 11.0, hemin catalyzed the H₂O₂-mediated oxidation of luminol to generate strong chemiluminescence signal; thus the target molecule itself served as an indicator for the molecule–aptamer interaction, which made the labeling and/or modification of aptamers or target molecules unnecessary. This label-free method for molecular recognition and DNA detection is therefore simple, easy, and effective. Figure A label-free approach to aptamer-based hemin recognition and DNA detection is introduced, which gives great potential for using a small molecule itself as the indicator for molecular recognition and DNA detection thereby avoiding any labeling or modification step     

Selective Hemin Binding by a Non-G-quadruplex Aptamer with Higher Affinity and Better Peroxidase-like Activity

Previous aptamers for porphyrins and metalloporphyrins were all guanine-rich sequences that can fold in G-quadruplex structures. Due to stacking-based binding, these aptamers can hardly tell different porphyrins apart, and they can also bind other planar molecules, hindering their practical applications. In this work, we used the capture selection method to obtain aptamers for hemin and protoporphyrin IX (PPIX). The hemin aptamer (Hem1) features two highly conserved repeating binding loops, and it cannot form a G-quadruplex, which was supported by its Mg²⁺-dependent but K⁺-independent hemin binding and CD spectroscopy. Isothermal titration calorimetry revealed much higher enthalpy change for the new aptamer, and the best aptamer showed a K_d of 43 nM hemin. Hem1 can also enhance the peroxidase-like activity of hemin. This work demonstrates that aptamers have alternative ways to bind porphyrins allowing selective recognition of different porphyrins.     

Fibre optic fluorometric enzyme sensors for hydrogen peroxide and lactate,based on horseradish peroxidase and lactate oxidase

An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalysed oxidation of homovanillic acid is utilized to determine the concentration of H₂O₂. The membrane-bound enzyme is attached to a bifurcated fibre bundle permitting excitation and detection of the fluorescence by a fluorometer. The response of the sensor is linear from 1 to 130 M hydrogen peroxide; the coefficient of variation is 3%. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 M and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum.     

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern

We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one-round SELEX experiments using five different trimeric spike proteins from variants, followed by high-throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed K_d values ranging from 2 to 10 nM for all variant spike proteins, and also bound similarly to variants not present in the reselection experiments. This aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with K_d values between 20 and 50 pM, and was integrated into a simple colorimetric assay for detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of a single protein, including emerging variants, making it well-suited for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.     

Novel electron transfer mediators based on dichloroindophenol derivatives for lactate oxidase

Novel indophenol derivatives were synthesized and characterized electrochemically with respect to their abilities to act as electron-transfer mediators for lactate oxidase. These compounds showed suitable redox potentials and high second-order rate constants, k_med, in solution for electron-transfer from the reduced enzyme. A lactate sensor using these derivatives demonstrated high sensitivity and good substrate selectivity. This sensor could also achieve excellent durability which retained more than 70% residual activity and good linearity in the range from 0 to 16 mM lactate concentration even after 10 days.     

Flow injection analysis of L-lactic acid using an enzyme-polyion complex-coated electrode as the detector

The concentration of L-lactic acid was determined by a combination of flow injection analysis with amperometric enzyme sensor detection. The enzyme sensor was prepared by immobilizing lactate oxidase in a layer of polyion complex consisting of poly-L-lysine and poly(4-styrenesulfonate). The sensor-based system can be used for the determination of L-lactate concentration up to 6 mM with a sampling rate of 120 h(-1), and is stable for 8 weeks after 1000 L-lactate injections. The permselectivity of the polyion complex matrix is effective for reducing the response from electrochemical interferents such as L-ascorbic acid, uric acid and acetaminophen.     

Alcohol,lactate and glutamate sensors based on oxidoreductases with regeneration of nicotinamide adenine dinucleotide

Flowthrough enzyme electrodes are reported for determinations of alcohol, lactate and glutamate. Oxidoreductases mixed with immobilized NAD⁺ cofactor are held between a suitable platinum electrode and a semipermeable membrane. The coenzyme is readily regenerated either directly by electrochemical oxidation or by using phenazine methosulphate (PMS⁺) as intermediate. Continuous flow conditions are used. The sensitivity obtained with the alcohol dehydrogenase electrode was 50, 620 or 810 nA mol^-1 of ethanol, respectively, when regeneration was done electrochemically or with 0.1 or 0.5 mM PMS⁺. The sensitivities for the lactate and glutamate sensors in the presence of 0.5 mM PMS⁺, were 14 and 50 nA mmol^-1 for D,L-lactate and L-glutamate, respectively. The calibration curves were linear for concentrations up to 0.5, 1.5 and 100 mM of glutamate, lactate and ethanol, respectively. The sensitivity of the alcohol and lactate sensors decreased by 50–55% within 60 h and that of the glutamate sensor within 6 h.     

Selection and identification of ssDNA aptamers recognizing zearalenone

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum on maize and barley. Because most current methods of ZEN detection rely on the use of low-stability antibodies or expensive equipment, we sought to develop a rapid, low-cost determination method using aptamers instead of antibodies as the specific recognition ligands. This work describes the isolation and identification of single-stranded DNA (ssDNA) aptamers recognizing ZEN using the modified systematic evolution of ligands by exponential enrichment methodology based on magnetic beads. After 14 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 12 representative sequences were assayed for their affinity and specificity. The best aptamer, 8Z₃₁, with a dissociation constant (K _d) of 41?±?5 nM, was successfully applied in the specific detection of ZEN in binding buffer and in real samples based on a magnetic separation/preconcentration procedure. This analytical method provided a linear range from 3.14?×?10^?9 to 3.14?×?10^?5 M for ZEN, and the detection limit was 7.85?×?10^?10 M. The selected aptamers are expected to be used in the potential development of affinity columns, biosensors, or other analytical systems for the determination of ZEN in food and agricultural products.

A microband lactate biosensor fabricated using a water-based screen-printed carbon ink

The present study demonstrated for the first time that screen-printed carbon microband electrodes fabricated from water-based ink can readily detect H₂O₂ and that the same ink, with the addition of lactate oxidase, can be used to construct microband biosensors to measure lactate. These microband devices were fabricated by a simple cutting procedure using conventional sized screen-printed carbon electrodes (SPCEs) containing the electrocatalyst cobalt phthalocyanine (CoPC). These devices were characterised with H₂O₂ using several electrochemical techniques. Cyclic voltammograms were found to be sigmoidal; a current density value of 4.2 mA cm⁻² was obtained. A scan rate study revealed that the mass transport mechanism was a mixture of radial and planar diffusion. However, a further amperometric study under quiescent and hydrodynamic conditions indicated that radial diffusion predominated. A chronoamperometric study indicated that steady-state currents were obtained with these devices for a variety of H₂O₂ concentrations and that the currents were proportional to the analyte concentration. Lactate microband biosensors were then fabricated by incorporating lactate oxidase into the water-based formulation prior to printing and then cutting as described. Voltammograms demonstrated that lactate oxidase did not compromise the integrity of the electrode for H₂O₂ detection. A potential of +400 mV was selected for a calibration study, which showed that lactate could be measured over a dynamic range of 1-10 mM which was linear up to 6 mM; a calculated lower limit of detection of 289 μM was ascertained. This study provides a platform for monitoring cell metabolism in-vitro by measuring lactate electrochemically via a microband biosensor.     

Magnetic relaxation switch and colorimetric detection of thrombin using aptamer-functionalized gold-coated iron oxide nanoparticles

We describe a sensitive biosensing system combining magnetic relaxation switch diagnosis and colorimetric detection of human α-thrombin, based on the aptamer–protein interaction induced aggregation of Fe₃O₄@Au nanoparticles. To demonstrate the concept, gold-coated iron oxide nanoparticle was synthesized by iterative reduction of HAuCl₄ onto the dextran-coated Fe₃O₄ nanoparticles. The resulting core–shell structure had a flowerlike shape with pretty narrow size distribution (referred to as “nanorose”). The two aptamers corresponding to human α-thrombin were conjugated separately to two distinct nanorose populations. Once a solution containing human α-thrombin was introduced, the nanoroses switched from a well dispersed state to an aggregated one, leading to a change in the spin–spin relaxation time (T₂) as well as the UV–Vis absorption spectra of the solution. Thus the qualitative and quantitative detection method for human α-thrombin was established. The dual-mode detection is clearly advantageous in obtaining a more reliable result; the detection range is widened as well. By using the dual-mode detection method, a detectable T₂ change is observed with 1.0 nM human α-thrombin, and the detection range is from 1.6 nM to 30.4 nM.     

Development of an Enzymeless/Mediatorless Glucose Sensor Using Ruthenium Oxide‐Prussian Blue Combinative Analogue

Presented in this work is the first step towards an enzymeless/mediatorless glucose sensor. We first observed remarkable electrocatalytic oxidation of glucose using combinative ruthenium oxide (RuOx)‐Prussian blue (PB) analogues (designated as mvRuOx‐RuCN, mv: mixed valent) at ca. 1.1 V (vs. Ag/AgCl) in acidic media (pH 2 Na₂SO₄/H₂SO₄). Individual RuOx and PB analogs failed to give any such catalytic response. A high ruthenium oxidation state (i.e., oxy/hydroxy‐Ru^VII, E°≈1.4 V vs. RHE), normally occurring in strong alkaline conditions at RuOx‐based electrodes, was electrogenerated and stabilized (without any conventional disproportionation reaction) in the mvRuOx‐RuCN matrix for glucose catalysis. Detail X‐ray photoelectron spectroscopic studies can fully support the observation. The catalyst was chemically modified onto a disposable screen‐printed carbon electrode and employed for the amperometric detection of glucose via flow injection analysis (FIA). This system has a linear detection range of 0.3–20 mM with a detection limit and sensitivity of 40 μM (S/N=3) and 6.2 μA/(mM cm²), respectively, for glucose. Further steps towards the elimination of interference and the extendibility to neutral pHs were addressed.     

Effect of methyltin trichloride on the activity of lactate dehydrogenase

The effect of MeSnCl₃, which is a highly toxic compound, on the activity of L-lactate:NAD oxidoreductase (lactate dehydrogenase) in the extract from the liver of Russian sturgeon (Asipenser gueldenstaedtiB.). Noncompetitive inhibition of the enzymatic reaction was discovered. This can be due to a change in the enzyme conformation caused by the action on the thiol groups, important for enzyme activity.     

Novel CeO2–CuO-decorated enzymatic lactate biosensors operating in low oxygen environments

The detection of the lactate level in blood plays a key role in diagnosis of some pathological conditions including cardiogenic or endotoxic shocks, respiratory failure, liver disease, systemic disorders, renal failure, and tissue hypoxia. Here, we described for the first time the use of a novel mixed metal oxide solution system to address the oxygen dependence challenge of first generation amperometric lactate biosensors. The biosensors were constructed using ceria-copper oxide (CeO₂–CuO) mixed metal oxide nanoparticles for lactate oxidase immobilization and as electrode material. The oxygen storage capacity (OSC, 492 μmol-O₂/g) of these metal oxides has the potential to reduce the oxygen dependency, and thus eliminate false results originated from the fluctuations in the oxygen concentration. In an effort to compare the performance of our novel sensor design, ceria nanoparticle decorated lactate sensors were also constructed. The enzymatic activity of the sensors were tested in oxygen-rich and oxygen-lean solutions. Our results showed that the OSC of the electrode material has a big influence on the activity of the biosensors in oxygen-lean environments. While the CeO₂ containing biosensor showed an almost 21% decrease in the sensitivity in a O₂-depleted solution, the CeO₂–CuO containing electrode, with a higher OSC value, experienced no drop in sensitivity when moving from oxygen-rich to oxygen-lean conditions. The CeO₂–CuO decorated sensor showed a high sensitivity (89.3 ± 4 μA mM⁻¹ cm⁻²), a wide linear range up to 0.6 mM, and a low limit of detection of 3.3 μM. The analytical response of the CeO₂–CuO decorated sensors was studied by detecting lactate in human serum with good selectivity and reliability. The results revealed that CeO₂–CuO containing sensors are promising candidates for continuous lactate detection.     

Aptamer-based detection of thrombin by acoustic method using DNA tetrahedrons as immobilisation platform

The thickness shear mode acoustic method was used to study the binding of thrombin to DNA aptamers immobilised on the gold surface covered by DNA tetrahedrons. The binding of thrombin to conventional aptamers sensitive to fibrinogen (FBT) and heparin (HPT) exosites as well as to HPT in a loop configuration (HPTloop) made it possible to determine the constant of dissociation (K_D) and the limit of detection (LOD). The sensing system composed of a HPTloop was characterised by K_D = (66.7 ± 22.7) nM, which was almost twice as low as that of FBT and HPT. For this biosensor, a lower LOD of 5.2 nM compared with 17 nM for conventional HPT aptamers was also obtained. Less sensitive sensors based on FBT aptamers revealed an LOD of 30 nM which is in agreement with the lower affinity of these aptamers to thrombin in comparison with that of HPT. The surface concentration of DNA tetrahedrons was determined by the electrochemical method using [Ru(NH₃)₆]³⁺ as a redox probe. These experiments confirmed that the “step by step” method of forming the sensing layer, consisting first in chemisorption of DNA tetrahedrons onto a gold surface and then in hybridisation of the aptamer-supporting part with complementary oligos at the top of the tetrahedron, is preferable. In addition, atomic force microscopy was applied to analyse the topography of the gold layers modified stepwise by DNA tetrahedrons, DNA aptamers and thrombin. The height profiles of the layers were in reasonable agreement with the dimensions of the adsorbed molecules. The results indicate that DNA tetrahedrons represent an efficient platform for immobilisation of aptamers.     

Colorimetric method for determination of bisphenol A based on aptamer-mediated aggregation of positively charged gold nanoparticles

A sensitive, specific and rapid colorimetric aptasensor for the determination of the plasticizer bisphenol A (BPA) was developed. It is based on the use of gold nanoparticles (AuNPs) that are positively charged due to the modification with cysteamine which is cationic at near-neutral pH values. If aptamers are added to such AuNPs, aggregation occurs due to electrostatic interactions between the negatively-charged aptamers and the positively-charged AuNPs. This results in a color change of the AuNPs from red to blue. If a sample containing BPA is added to the anti-BPA aptamers, the anti-BPA aptamers undergo folding via an induced-fit binding mechanism. This is accompanied by a conformational change, which prevents the aptamer-induced aggregation and color change of AuNPs. The effect was exploited to design a colorimetric assay for BPA. Under optimum conditions, the absorbance ratio of A ₅₂₇/A ₆₈₀ is linearly proportional to the BPA concentration in the range from 35 to 140 ng∙mL⁻¹, with a detection limit of 0.11 ng∙mL⁻¹. The method has been successfully applied to the determination of BPA in spiked tap water and gave recoveries between 91 and 106 %. Data were in full accordance with results obtained from HPLC. This assay is selective, easily performed, and in our perception represents a promising alternative to existing methods for rapid quantification of BPA.

The negatively-charged anti-BPA aptamers can absorb onto the positively-charged cysteamine-capped AuNPs (cysteamine-AuNPs) via electrostatic interactions, which can cause the aggregation of AuNPs accompanied by a red-to-blue color change. In the presence of BPA, the specific binding of BPA to the aptamers induces the conformation changes of anti-BPA aptamers, which can release the aptamers from cysteamine-AuNPs and thus prevent the aggregation and color change of cysteamine-AuNPs.

     

Highly Specific Recognition of Guanosine Using Engineered Base-Excised Aptamers

Purines and their derivatives are highly important molecules in biology for nucleic acid synthesis, energy storage, and signaling. Although many DNA aptamers have been obtained for binding adenine derivatives such as adenosine, adenosine monophosphate, and adenosine triphosphate, success for the specific binding of guanosine has been limited. Instead of performing new aptamer selections, we report herein a base-excision strategy to engineer existing aptamers to bind guanosine. Both a Na⁺-binding aptamer and the classical adenosine aptamer have been manipulated as base-excising scaffolds. A total of seven guanosine aptamers were designed, of which the G16-deleted Na⁺ aptamer showed the highest bindng specificity and affinity for guanosine with an apparent dissociation constant of 0.78 mm . Single monophosphate difference in the target molecule was also recognizable. The generality of both the aptamer scaffold and excised site were systematically studied. Overall, this work provides a few guanosine binding aptamers by using a non-SELEX method. It also provides deeper insights into the engineering of aptamers for molecular recognition.