Multifunctional Label-Free Electrochemical Biosensor Based on an Integrated Aptamer

A novel AIEgen-Based probe Integrated with split aptamer Conjugates for adenosine triphosphate detection in beer.

Adenosine triphosphate (ATP) is crucial for cellular activity. The need for ATP detection in the field of biomedicine is rapidly increasing. Several biosensor-based approaches have been developed as a result of the growing demand for ATP detection. An electrolyte-insulator-semiconductor (EIS) sensor is a type of field-effect device that has the ability to detect surface-potential changes with a specific level of sensitivity. In this study, a label-free ATP detection biosensor based on poly(amidoamine)-modified EIS sensors was developed, in which an ATP-sensitive aptamer (Apt) was utilized as the sensitive element and the EIS sensor was used as the transducer. It is possible to monitor the binding of charged molecules, such as aptamers using EIS sensors in a label-free manner with a straightforward setup. To improve the coupling efficiency of Apt with the EIS sensor, a positively charged polyelectrolyte, i.e., poly (amidoamine) (PAMAM) dendrimers, was utilized to modify the surface of the EIS sensors to attach the negatively charged Apt through electrostatic attraction. The adsorptive binding of Apt and ATP results in a change in the capacitance of the EIS sensor. During the process of surface modification, the electrochemical measurements of capacitance-voltage (C-V) curves and constant-capacitance (ConCap) characteristics were utilized as indicators for the corresponding processes of EIS sensor surface modification. The measurement results indicated that this biosensor was able to detect ATP with high sensitivity and good specificity. The detection range of ATP was from 0.1 nM to 100 nM and the detection limit was as low as 0.12 nM. This biosensor has the potential to be utilized in the detection of ATP in the surrounding microenvironment of cells and tissues, with promising prospects for application in the field of biomedicine such as energy and metabolism monitoring of cells and tissues.

Aptamer-conjugated silver nanoparticles for electrochemical detection of adenosine triphosphate

A new electrochemical aptasensor based on electrocatalytic property of graphene toward ascorbic acid oxidation

Graphene foam field-effect transistor for ultra-sensitive label-free detection of ATP

A microfluidic device for adenosine triphosphate (ATP) was checking of fish freshness. Sample solutions containing ATP and the other solution containing an enzyme substrate were handled in the form of plugs formed in a network of flow channels. To simplify the structure and procedure for mixing solutions, a wider portion was formed in a flow channel to merge the plugs. A row of plugs were ejected from each of three branched flow channels, merged at the wider portion, and transported to an ATP biosensor located in the lower stream. ATP was detected using two enzymatic reactions involving glycerol kinase (GK) and glycerol-3-phosphate oxidase (G3PO). The current observed with solutions of three different ATP concentrations showed different values, demonstrating that the device can be used for multi-point sensing. Furthermore, the detection of ATP in extracts from tuna was carried out and a distinct increase in current was observed as in the case of standard solutions.

Antifouling aptasensor for the detection of adenosine triphosphate in biological media based on mixed self-assembled aptamer and zwitterionic peptide

Pathogens such as porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome (PRRSV), and E. coli are known to spread by contaminated vehicles and equipment. Pork producers have adopted trailer wash policies where each trailer is washed, disinfected, and dried before it can return to a farm. Cleanliness of livestock trailers after washing is determined by visual inspection rather than any objective method. Adenosine triphosphate (ATP) bioluminescence is used in many industries to provide real-time feedback on surface cleanliness through the detection of ATP from organic sources. That same technology may provide trailer wash facilities a way of objectively characterizing a livestock trailer's suitability to return to a farm after washing. Two ATP luminometers (3M Clean-Trace and Neogen AccuPoint) were used to estimate the correlation between ATP bioluminescence readings and aerobic bacterial plate counts (APCs) from sampled surfaces and to determine locations within a livestock trailer that can accurately estimate surface cleanliness. Five locations in livestock trailers were evaluated. Those locations included the nose access door (NAD), back door flush gate, rear side access door (RSAD), belly flush gate (BFG), and belly side access door (BSAD). There was a positive log-log association between the two luminometers (r = 0.59, P < 0.01). Every log unit increase in one unit, resulted in a 0.42 log increase (P < 0.01) in the other unit. ATP can come from bacteria, yeasts, molds, and manure. There was a poor association (r ≥ 0.10, P ≥ 0.02) between APCs and the ATP luminometers. Still, an increase in relative light units (RLUs) resulted in a corresponding increase in colony-forming units. The greatest area of surface contamination measured by APC was the NAD. RLUs were also greater in the NAD compared to the RSAD, the BFG, and the BSAD (P ≤ 0.01). Because APCs and luminometer RLUs provided similar outcomes, statistical process control charts were developed to determine control limits for RLUs. This provides real-time feedback to trailer wash workers in determining cleanliness outcomes for livestock trailers. These data suggest that ATP bioluminescence can be a reliable method to monitor cleaning effectiveness in livestock trailers. Bioluminescence is a monitoring tool that should be used in conjunction with microbial methods to monitor procedures for cleaning and disinfection.

A molecularly imprinted polymer (MIP) based capacitive sensor for antibiotic detection in drinking water and milk has been developed on a gold coated silicon electrode (Au Electrode). The electrode was fabricated by electropolymerizing monomer resorcinol (RN) on Au surface in presence of sulphanilamide (SN) as a template molecule, to get insulated RN polymer antibiotic composite. The insulation of the polymer film was improved by incubation of electrode in 1‐Dodecanethiol solution. Subsequently MIP sensor was obtained by extraction of SN in ethanol and acetic acid solution. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measurements were performed for characterization of the developed MIP electrode at different steps of fabrication. The surface morphology of MIP electrode was characterized using atomic force microscopy (AFM), X‐ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive x‐ray spectroscopy (EDS). Performance of MIP sensor was evaluated by measuring change in capacitance against varying concentration of SN using EIS. A linear response in the range 1 to 200 μg L−1 SN was recorded for MIP sensor with a detection limit of 0.1 μg L−1. The developed MIP sensor exhibited good selectivity towards SN in water and milk with recoveries in the range 92 % to 105 %. The obtained results suggest the usability of MIP based sensor for SN estimation in water and milk samples.

Adenosine triphosphate (ATP) is a critical intracellular energy currency that plays a key role in various cellular processes and is closely associated with numerous diseases. Similarly, biothiols such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are integral to many physiological and pathological processes due to their strong redox properties. Simultaneous discrimination and detection of ATP and biothiols offer valuable insights into the pathogenesis of conditions such as epilepsy and liver injury. This study introduces the first fluorescent probe, BCR, designed for multifunctional detection of ATP, GSH, Hcy, and Cys. With outstanding optical properties, excellent biocompatibility, high selectivity, and superior sensitivity, probe BCR enables effective imaging of ATP and biothiol dynamics in vivo. Moreover, probe BCR successfully visualizes changes in ATP, GSH, Hcy, and Cys levels in a PTZ‐induced epileptic zebrafish model and an APAP‐induced mouse liver injury tissue section model. These findings underscore the significant potential of probe BCR for early disease diagnosis and therapeutic applications.

There are many reasons for testing cell viability. Simple tinctorial assays, such as trypan blue exclusion, have their place, but luminescence assays based on the detection of adenosine triphosphate (ATP) are particularly useful, since it is possible to detect the ATP present in fewer than 20 cells/mL using optimized reagents. These same reagents can also accurately determine the ATP from 2 × 107 cells/mL in an adjacent well or test tube with a completely linear relationship between cell number and light output, provided that the cells maintain a relatively invariant ATP content. Similar methods are used for bacteria and eukaryotic cells, but this chapter is restricted to the consideration of eukaryotic cells. Luminescence measurement of ATP levels uses the following reaction:KeywordsLymphocyte Transformation TestTrypan Blue SolutionAutomate PipetUniversal TubeMaximum Nontoxic ConcentrationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Aptamer-based strip assay is an easy, highly efficient and low-cost detection method, which has been developed and easily applied to onsite detection. A new sensitive sandwich dipstick assay for adenosine triphosphate (ATP) detection was successfully developed based on specific recognition between split aptamer fragments and the target. In this method, the thiolated aptamer was first conjugated to the surface of gold nanoparticles (AuNPs), while the biotin-aptamer was immobilized on the surface of a nitrocellulose filter in the test line. In the presence of ATP, the thiol-aptamer/ATP/biotin-aptamer complexes were generated, which led to an obvious increase in optical signals at the test line. Under the optimal determination conditions, an excellent linear logarithmic response to the ATP concentration was obtained within the range of 0.5μM to 5mM. The limit of detection (LOD) of 0.5μM was reached at a signal-to-noise ratio of 3. The dipstick assay showed a good average recovery of 96-108% with the RSD of less than 20% in urine samples. The proposed method exhibited high specificity against other nucleotides such as the uridine triphosphate (UTP), cytidine triphosphate (CTP), and guanosine triphosphate (GTP). The results indicated that the dipstick strip may be considered as an inexpensive screening tool for onsite ATP determination. Graphical Abstract A simple split aptamer fragments based sandwich-type dipstick assay was developed for ATP detection.

The University of Michigan Risk Management Department recently took a close look at medical device reprocessing, inspired by the U.S. Food and Drug Administration's interest in this area. This study aimed to discover if the current cleaning methods were effective, identify contaminants left behind, and identify ways of making medical device processing more efficient and affordable.Testing began at the University of Michigan Health System in the Central Sterile Processing Department (CSPD) at a workstation specifically designed and assembled for this task. The workstation was equipped with sinks, brushes and enzymatic cleaners appropriate for safe handling of medical devices. Unique additions included a new automated reprocessor featuring cycles of mechanical and ultrasonic cleaning, a borescope, and a digital camera system for inspecting and recording lumen interiors, as well as extra workspace and supplies for the collection and chemical analysis of residues extracted from the medical devices.From internal review, many devices were known to be hard to clean, such as septorhinoplasty sets, due to the cannulae, and coronary suctions, due to cannula shape. A variety of these were tested early on to obtain a working data set to establish the scope of the problem (Table 1). The focus soon turned to suctions (Figure 1), which were ideal for study due to the many variations of design, soils they come into contact with, and the very large number of individual suctions available in CSPD at any given time.It is axiomatic in the industry that medical devices that cannot be cleaned cannot be considered sterile. So one cannot assume that all fully reprocessed devices are sterile. Practical considerations create acceptance of the standards: Reusable surgical instruments are a requirement, as are a steady supply and quick turn-around time to supply clinical needs. An additional challenge is the wide variety of devices used, with broad variations in size, shape, composition, and use. A device designed to pinch, pull, or hold during surgery will also do so in reprocessing. The challenge lies in finding a way to ensure the debris held by devices is removed.The unique characteristics of a specific surgical device can provide a great challenge to cleaning and the verification of effective cleaning. Because many of the critical surfaces of a device are difficult or impossible to visually inspect, other cleaning verification methods are needed. In addition, there are critical limitations to cleaning verification in an CSPD, including lack of laboratory equipment, lack of staff trained as laboratory technicians, and limited time for testing. The need then, is for tests that provide very rapid results (within minutes), are simple to use, and simple to interpret (for example, with pass-fail results).Such tests are already commercially available, and address these issues to varying degrees. Typically these tests involve either swabbing an instrument, or flushing water through the channel(s) of the instrument, to extract any residual soil that may remain for testing. Tests for cleanliness use markers likely to be found in any bodily secretion, such as protein or adenosine triphosphate (ATP); or markers that are secretion-specific, such as hemoglobin in blood. Two questions arise, however: What is the soil(s) that is being tested for, and is the method used to extract the soil effective? In our experience with suction tips, the greater challenge has been the efficacy of the extraction method, which makes identifying the soil more difficult.Most organic soils in the body are water soluble. However, when exposed over time to heat and chemicals, these organic soils can be rendered insoluble. This is particularly true when exposed to the high temperatures of steam sterilization. In these conditions, organic soils, including proteins, denature and can become fixed on the device surface. After repeated sterilizations, these soils can become almost “fused” with the device substrate, including stainless steel. The result, as we have observed, is obvious staining of the device with soil that is not readily or reliably extracted using nondestructive means such as swabbing or flushing. Solidified organic debris can flake off during use, posing an immediate risk to the patient. It can also harbor microbial contamination, another possible risk to the patient.The issue of “what is clean” is a well-discussed but not well-defined topic. In theory, clean means the complete absence of all contaminants, both organic and inorganic. In the real world, however, it is not possible to completely remove all contamination. Even if it were, it is not possible to confirm 100% removal. To compromise, one has to select tests and test methods that can access the most difficult-to-clean areas of a device and detect clinically relevant soil(s) that would be expected to be present. Soils that could be expected include:Once the composition of the soil present is known, it is easier to select an appropriate test method. Most suction tips we focused on primarily came into contact with blood. It would be logical to expect that the reddish soil or apparent staining observed on the internal channel of these medical devices is dried blood. There are readily available kits for blood testing (such as a peroxide reaction with hemoglobin), which are highly sensitive (detect 0.1μg), rapid (result in 30 seconds), and easy to interpret (produce dramatic color change).These tests also lend themselves to either a swab or flush extraction method. Unfortunately, as we have seen, soil observed inside these devices can be highly insoluble, and often cannot be harvested by either swabbing or flushing. Theoretically, more aggressive means could be employed to extract these soils, but could mean damage to the device (from scraping), or use of unsafe chemical solvents.Direct inspection of suction devices and other instruments with a flexible fiber optic bore-scope revealed widespread staining and debris in a variety of used devices that had been terminally processed (ready for use), including a suction tip (Figure 2) and an arthroscopic shaver (Figures 3-4). Stains range in color from red to black. Intuitively, the red might be blood. The black could also be blood, other proteins, build-up of detergents, or oxidation of the steel.To determine the composition of the stains, we conducted a variety of tests. The objective was to identify a test or panel of tests that could reliably detect the staining without destruction of the device, and without the use of a bore-scope. Of course, the composition of the stain would also indicate the source of contamination, and the potential risk to patients.The nondestructive tests we selected were hemoglobin (peroxidase reaction), protein (pyromol and bromophenol blue reactions), as well as ATP and bacterial detection, utilizing an enzyme reaction. Testing was conducted several times, over a period of several months, and each test panel included hemoglobin, protein, and ATP testing. A fourth test, bacterial detection by enzyme reagent, was added in later panels as a method for detecting possible biofilm formation. Extraction methods included flushing and swabbing over the series of tests. Visual inspection of the lumened devices was also conducted utilizing a borescope.Overall, while each method tested demonstrated positive results—with hemoglobin the most effective of the methods used—frequently, none of the tests proved to be positive for residuals, even when borescope inspection of the device revealed staining. It is likely that the reason for this is that the flushing and/or swabbing did not extract sufficient material. It is also possible that the staining was not due to organic soils, but some other source, such as oxidation of the steel.Given the difficulty of extracting soils for testing, destructive testing was considered. Use of surface analysis techniques offered an attractive option to identify soil composition. As an initial test, an instrument that had obvious staining, a Yankauer suction, was chosen for destruction (similar to the instrument in Figure 1). The first round of testing via scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX), showed that the red stains in the device were comprised of organic compounds. (See the sidebar on page 52 for more on these techniques.)On this individual device, further testing via Fourier transform infrared spectroscopy (FTIR) showed that the staining was a combination of iron oxide, hydrocarbons, and esters, which could be indicative of a biological source such as blood and fatty acids, or a non-biological source such as steel oxidation and lubricating oils. Further analysis via X-ray photoelectron spectroscopy (XPS) was positive for organic compounds consistent with bodily secretions, but other sources are also possible. As this was one test on one device, the results cannot be generalized. A much larger study is required to obtain a good data set of stain composition.As the nondestructive testing discussed earlier revealed, the source of staining may often be bodily fluids. A study, which involves dozens or perhaps hundreds of clinically used devices subjected to destructive testing, is required to develop an evidentiary-based conclusion as to which soils are the sources of stains in these devices. Such an undertaking could be expensive and time consuming, but well worth the effort for an industry seeking to improve the reprocessing of devices used for patient care.The discovery of a problem is only the first step, but a step in the right direction. The testing procedures described here can be repeated in any CSPD or surgical center, using any of the medical devices and tools described. The questions raised are: Are the industry standards for device reprocessing adequate? Do instruments that cannot be cleaned pose a risk to patients? How can the cleaning process be improved?The next step proposed is continued and focused testing. Having identified the necessary steps, continued data collection is paramount, as this information will impact medical device reprocessing specifications and requirements.

The cationic water-soluble poly(diethynylfluorene-co-diethynylcarbazole-co-diethynylbenzothiadiazole) (P1–P3) were synthesized and used for adenosine triphosphate (ATP) detection. The incorporation of diethynylbenzothiadiazole (DBT) into the polymer backbone offers dual emissive polyelectrolytes with both blue and red emission bands in the dilute solution resulting from incomplete intramolecular energy transfer. In the solution of P2, addition of ATP can quench the emission of fluorene-co-carbazole units of P2 more efficiently than that of DBT segments, which causes the fluorescent color change from blue to red. The asymmetric quenching of the blue and red emission bands in the presence of ATP might become a possible method for a real-time ATP detection. In addition, when ATP is cleaved into fragments by enzyme alkaline phosphatase (ALP), the blue emission of P2 can be recovered. Thus, it is possible to assay the enzyme activity by triggering the change in color and intensity of the emission of the solution of P2.

Visual inspections are used to assess hospital cleanliness, as visible contamination may present health risks and influence perceptions of care quality. Problematically, many contaminants are invisible to the naked eye, limiting the reliability of visual checks. Many invisible substances, however, fluoresce (i.e. emit visible light after absorbing electromagnetic radiation). Portable torches can detect fluorescent substances in situ, offering a potential method to enhance cleaning practices. This study has evaluated fluorescence as a tool for identifying general invisible contamination after hospital cleaning. Visibly clean surfaces in seven single-occupancy patient rooms and two six-bed wards across two National Health Service hospitals were examined using a portable high-intensity blue and ultraviolet light torch. Adenosine triphosphate (ATP) levels in fluorescent and non-fluorescent areas were taken as a recognized cleaning monitoring tool, and analysed statistically using Wilcoxon signed-rank tests. Fluorescent contamination that was invisible to the naked eye was found on every surface. ATP relative light unit (RLU) levels were significantly higher in fluorescent substances compared with non-fluorescent substances (P≤0.05) with large effect sizes on patient chairs, bed frames, overbed tables, bedside units and pillows, but not toilets, sinks or commodes. The mean RLU measurement was 161 in fluorescent areas compared with 33 RLU in control areas. Fluorescence detected alternative contamination which could present toxic risk to humans, such as cleaning fluid and/or drug-contaminated residue which frequently contain fluorescent constituents. This was an important finding as relying solely on ATP detection may overlook significant contamination risks. Further work to evaluate the method as a cleaning aid is encouraged.