Fluorescent and electrochemical detection of nuclease activity associated with Streptococcus pneumoniae using specific oligonucleotide probes.

Streptococcus pneumoniae (S. pneumoniae) represents a significant pathogenic threat, often responsible for community-acquired pneumonia with potentially life-threatening consequences if left untreated. This underscores the pressing clinical need for rapid and accurate detection of this harmful bacteria. In this study, we report the screening and discovery of a novel biomarker for S. pneumoniae detection. We used S. pneumoniae nucleases as biomarker and we have identified a specific oligonucleotide that works as substrate. This biomarker relies on a specific nuclease activity found on the bacterial membrane, forming the basis for the development of both fluorescence and electrochemical biosensors. We observed an exceptionally high sensitivity in the performance of the electrochemical biosensor, detecting as low as 102 CFU mL-1, whereas the fluorescence sensor demonstrated comparatively lower efficiency, with a detection limit of 106 CFU mL-1. Moreover, the specificity studies have demonstrated the biosensors' remarkable capacity to identify S. pneumoniae from other pathogenic bacteria. Significantly, both biosensors have demonstrated the ability to identify S. pneumoniae cultured from clinical samples, providing compelling evidence of the potential clinical utility of this innovative detection system.

Real-Time Biosensing Bacteria and Virus with Quartz Crystal Microbalance: Recent Advances, Opportunities, and Challenges.

Continuous monitoring of pathogens finds applications in environmental, medical, and food industry settings. Quartz crystal microbalance (QCM) is one of the promising methods for real-time detection of bacteria and viruses. QCM is a technology that utilizes piezoelectric principles to measure mass and is commonly used in detecting the mass of chemicals adhering to a surface. Due to its high sensitivity and rapid detection times, QCM biosensors have attracted considerable attention as a potential method for detecting infections early and tracking the course of diseases, making it a promising tool for global public health professionals in the fight against infectious diseases. This review first provides an overview of the QCM biosensing method, including its principle of operation, various recognition elements used in biosensor creation, and its limitations and then summarizes notable examples of QCM biosensors for pathogens, focusing on microfluidic magnetic separation techniques as a promising tool in the pretreatment of samples. The review explores the use of QCM sensors in detecting pathogens in various samples, such as food, wastewater, and biological samples. The review also discusses the use of magnetic nanoparticles for sample preparation in QCM biosensors and their integration into microfluidic devices for automated detection of pathogens and highlights the importance of accurate and sensitive detection methods for early diagnosis of infections and the need for point-of-care approaches to simplify and reduce the cost of operation.

Surface plasmon resonance aptasensor for soluble ICAM-1 protein in blood samples.

Intercellular Adhesion Molecule-1 (ICAM-1) is considered to be a cancer biomarker in the assessment of metastatic potential in patients and an early indicator of atherosclerosis. A labelless biosensor based on the surface plasmon resonance (SPR) signal from the specific affinity interaction of an aptamer and a soluble ICAM-1 protein was developed for blood samples. The developed aptasensor provided real-time information on the concentration of the ICAM-1 protein in blood when integrated to a purification step based on a magnetic pull-down separation. The SPR aptasensor was highly specific with a limit of detection of 1.4/0.2 ng ml-1, which was achieved through aptamer-functionalized silica-coated magnetic nanoparticles.

Surface plasmon resonance aptasensor for Brucella detection in milk.

A Surface Plasmon Resonance (SPR) aptasensor was developed for the detection of Brucella melitensis (B. melitensis) in milk samples. Brucellosis is a bacterial zoonotic disease with global distribution caused mostly by contaminated milk or their products. Aptamers recognizing B. melitensis were selected following a whole bacteria-SELEX procedure. Two aptamers were chosen for high affinity and high specificity. The high affinity aptamer (B70 aptamer) was immobilized on the surface of magnetic silica core-shell nanoparticles for initial purification of the target bacteria cells from milk matrix. Another aptamer, highly specific for B. melitensis cells (B46 aptamer), was used to prepare SPR sensor chips for sensitive determination of Brucella in eluted samples from magnetic purification since direct injection of milk samples to SPR sensor chips is known for a high background unspecific signal. Thus, we integrated a quick and efficient magnetic isolation step for subsequent instant detection of B. melitensis contamination in one ml of milk sample by SPR with a LOD value as low as 27 ± 11 cells.

Targeted mesoporous silica nanoparticles for improved inhibition of disinfectant resistant Listeria monocytogenes and lower environmental pollution.

Benzalkonium chloride (BAC) is a common ingredient of disinfectants used for industrial, medical, food safety and domestic applications. It is a common pollutant detected in surface and wastewaters to induce adverse effects on Human health as well as aquatic and terrestrial life forms. Since disinfectant use is essential in combatting against microorganisms, the best approach to reduce ecotoxicity level is to restrict BAC use. We report here that encapsulation of BAC in mesoporous silica nanoparticles can provide an efficient strategy for inhibition of microbial activity with lower than usual concentrations of disinfectants. As a proof-of-concept, Listeria monocytogenes was evaluated for minimum inhibitory concentration (MIC) of nanomaterial encapsulated BAC. Aptamer molecular gate structures provided a specific targeting of the disinfectant to Listeria cells, leading to high BAC concentrations around bacterial cells, but significantly reduced amounts in total. This strategy allowed to inhibition of BAC resistant Listeria strains with 8 times less the usual disinfectant dose. BAC encapsulated and aptamer functionalized silica nanoparticles (AptBACNP) effectively killed only target bacteria L. monocytogenes, but not the non-target cells, Staphylococcus aureus or Escherichia coli. AptBACNP was not cytotoxic to Human cells as determined by in vitro viability assays.

Antibiotic administration in targeted nanoparticles protects the faecal microbiota of mice.

Antibiotic therapy comes with disturbances on human microbiota, resulting in changes of bacterial communities and thus leading to well-established health problems. In this study, we demonstrated that targeted teicoplanin administration maintains the faecal microbiota composition undisturbed in a mouse model while reaching therapeutic improvements for S. aureus infection.

Inhibitory effects of aptamer targeted teicoplanin encapsulated PLGA nanoparticles for Staphylococcus aureus strains.

Emergence of resistance to traditional antibiotic treatments necessitates alternative delivery systems. Teicoplanin is a glycopeptide antibiotic used in the treatments of serious infections caused by Gram-positive bacteria, including Methicillin Resistant Staphylococcus aureus (MRSA). One strategy to keep up with antibiotic resistance development is to limit dose and amount during treatments. Targeted delivery systems of antibiotics have been suggested as a mechanism to slow-down the evolution of resistance and to increase efficiency of the antimicrobials on already resistant pathogens. In this study, we report teicoplanin delivery nanoparticles of Poly Lactic-co-Glycolic Acid (PLGA), which are functionalized with S. aureus specific aptamers. A 32-fold decrease in minimum inhibitory concentration (MIC) values of teicoplanin for S. aureus was demonstrated for susceptible strains and about 64-fold decline in MIC value was achieved for moderately resistant clinical isolates of MRSA upon teicoplanin treatment with aptamer-PLGA nanoparticles. Although teicoplanin delivery in PLGA nanoparticles without targeting demonstrated eightfold decrease in MIC of susceptible strains of S. aureus and S. epidermidis and twofold in MIC of resistant strains, the aptamer targeting specifically decreased MIC for S. aureus, but not for S. epidermidis. Therefore, aptamer-targeted PLGA delivery of antibiotic can be an attractive alternative to combat with some of the multi-drug resistant bacterial pathogens.

Fibrous polymer functionalized magnetic biocatalysts for improved performance.

Although enzymes are known for their excellent catalytic performance, industrial, medical or biotechnological applications should overcome some drawbacks like long-term stability under specific conditions of the application. Immobilized enzymes have offered advantages over soluble counterparts in many industrial and laboratory scale applications by increasing operational stability and reusability. When the immobilization matrix has magnetic properties, an additional advantage is obtained as simpler processing. Iron-based superparamagnetic nano-sized particles has large surface area for bio-compatible applications are especially in focus. Adding nanofibrous polymers to magnetic nanoparticles has been an excellent way to increase efficiency of biocatalyst immobilization by further increasing loading capacity. This chapter explains various magnetic enzyme-nanoparticles based preparations with potential for future industrial applications like invertase, lipase and as model studies and focus on the nanofibrous polymer brush grafting as a way to increase catalytic efficiency of magnetic nanoparticles.

Label-free lateral flow assay for Listeria monocytogenes by aptamer-gated release of signal molecules.

Lateral flow assay (LFA) type of biosensors have been popular due to cost-effectiveness and easy-interpretation for instant results, most common examples of applications being pregnancy tests, food safety or medical diagnostics. There are several examples of reports with high sensitivity, including pre-concentration of the sample by magnetic pull-down. However, sensitivity and direct detection designs with aptamers has been a limiting factor for developing aptamers-based LFA assays. In this study, we report a lateral flow design based on aptamer-gated silica nanoparticles to develop high sensitivity and direct bacterial assay by shifting aptamers-target interaction to conjugation pad. Aptamer-gated silica nanoparticles-based biosensors were reported for their high sensitivity, specificity and label-free detection for small molecules and whole cells. This label-free strategy for LFA can determine L. monocytogenes in minced chicken matrix at less than 5 min with a limit of detection (LOD) of 53 cells in one mL samples.

Genotyping of single nucleotide polymorphism by probe-gated silica nanoparticles.

The development of simple, reliable, and rapid approaches for molecular detection of common mutations is important for prevention and early diagnosis of genetic diseases, including Thalessemia. Oligonucleotide-gated mesoporous nanoparticles-based analysis is a new platform for mutation detection that has the advantages of sensitivity, rapidity, accuracy, and convenience. A specific mutation in ß-thalassemia, one of the most prevalent inherited diseases in several countries, was used as model disease in this study. An assay for detection of IVS110 point mutation (A > G reversion) was developed by designing probe-gated mesoporous silica nanoparticles (MCM-41) loaded with reporter fluorescein molecules. The silica nanoparticles were characterized by AFM, TEM and BET analysis for having 180 nm diameter and 2.83 nm pore size regular hexagonal shape. Amine group functionalized nanoparticles were analysed with FTIR technique. Mutated and normal sequence probe oligonucleotides)about 12.7 nmol per mg nanoparticles) were used to entrap reporter fluorescein molecules inside the pores and hybridization with single stranded DNA targets amplified by PCR gave different fluorescent signals for mutated targets. Samples from IVS110 mutated and normal patients resulted in statistically significant differences when the assay procedure were applied.

Pathogen detection in complex samples by quartz crystal microbalance sensor coupled to aptamer functionalized core-shell type magnetic separation.

A quartz crystal microbalance sensor (QCM) was developed for sensitive and specific detection of Salmonella enterica serovar typhimurium cells in food samples by integrating a magnetic bead purification system. Although many sensor formats based on bioaffinity agents have been developed for sensitive and specific detection of bacterial cells, the development of robust sensor applications for food samples remained a challenging issue. A viable strategy would be to integrate QCM to a pre-purification system. Here, we report a novel and sensitive high throughput strategy which combines an aptamer-based magnetic separation system for rapid enrichment of target pathogens and a QCM analysis for specific and real-time monitoring. As a proof-of-concept study, the integration of Salmonella binding aptamer immobilized magnetic beads to the aptamer-based QCM system was reported in order to develop a method for selective detection of Salmonella. Since our magnetic separation system can efficiently capture cells in a relatively short processing time (less than 10 min), feeding captured bacteria to a QCM flow cell system showed specific detection of Salmonella cells at 100 CFU mL(-1) from model food sample (i.e., milk). Subsequent treatment of the QCM crystal surface with NaOH solution regenerated the aptamer-sensor allowing each crystal to be used several times.

NanoKeepers: stimuli responsive nanocapsules for programmed specific targeting and drug delivery.

Bacterial resistance is a high priority clinical issue worldwide. Thus, an effective system that rapidly provides specific treatment for bacterial infections using controlled dose release remains an unmet clinical need. Herein, we report on the NanoKeepers approach for the specific targeting of S. aureus with controlled release of antibiotics based on nuclease activity.

An experimental study of the regulation of glycolytic oscillations in yeast.

We have studied oscillating glycolysis in the strain BY4743 and isogenic strains with deletions of genes encoding enzymes in glycolysis, mitochondrial electron transport and ATP synthesis. We found that deletion of the gene encoding the hexokinase 1 isoform does not affect the oscillations while deletion of the gene encoding the hexokinase 2 isoform results in oscillations with smaller amplitude. The latter is associated with an almost 50% decrease in hexokinase activity. Deletions in the genes encoding the α- and ß-subunits of phosphofructokinase abolish the oscillations entirely. This loss in oscillatory activity is associated with a fourfold decrease in phosphofructokinase activity. Deletions of genes encoding subunits of the F1F0 ATPase also inhibit the oscillations in accordance with earlier studies using for example inhibitors. Finally, we identified an apparently new control point involving the mitochondrial cytochrome c oxidase. The latter is difficult to explain as oscillatory activity entails 100% inhibition of this enzyme. The mitochondria of this strain seem to have normal F1F0 ATPase activity. Overall these results support earlier experimental and model studies suggesting that in addition to processes within glycolysis also processes outside this pathway contribute to the control of the oscillatory behaviour.

Nanoparticle embedded enzymes for improved lateral flow sensors.

In this study, combining the nanoparticle embedded sensors with lateral flow assays, a novel strategy for ensuring the quality of signalling in lateral flow assays (LFAs) was developed. A LFA for reactive oxygen species (ROS) is reported that is based on horse radish peroxidase (HRP) which is co-entrapped with Texas Red dextran inside porous polyacrylamide nanoparticles. In this system, enzymes are protected in the porous matrix of polyacrylamide which freely allows the diffusion of the analyte. The sensor is rapid and sensitive for quantification of hydrogen peroxide concentrations. A test solution of hydrogen peroxides was quantified with this novel LFA-ROS sensor to obtain a linear range between 1 and 25 µM. Nanoparticle embedding of enzymes is proposed here as a general strategy for developing enzyme-based lateral flow assays, eliminating adverse effects associated with biological samples.

Targeting cancer cells with controlled release nanocapsules based on a single aptamer.

Molecular gates have received considerable attention as drug delivery systems. More recently, aptamer-based gates showed great potential in overcoming major challenges associated with drug delivery by means of nanocapsules. Based on a switchable aptamer nanovalves approach, we herein report the first demonstration of an engineered single molecular gate that directs nanoparticles to cancer cells and subsequently delivers the payload in a controllable fashion.

Characterization of structural changes in aptamer films for controlled release nanodevices.

The dimension of the conformational changes of DNA-aptamers which can be used as stimulus-responsive gate-keepers in controlled delivery nanodevices has been determined by acoustic wave-based sensors upon molecular recognition of a small-sized target, adenosine-5'-monophosphate (AMP).

Time-resolved measurements of intracellular ATP in the yeast Saccharomyces cerevisiae using a new type of nanobiosensor.

Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F(0)F(1) ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.

Aptamers embedded in polyacrylamide nanoparticles: a tool for in vivo metabolite sensing.

We describe a new type of aptamer-based optical nanosensor which uses the embedding of target responsive oligonucleotides in porous polyacrylamide nanoparticles to eliminate nuclease instability. The latter is a common problem in the use of aptamer sensors in biological environments. These aptamers embedded in nanoparticles (AptaNPs) are proposed as a tool in real-time metabolite measurements in living cells. The AptaNPs comprise 30 nm polyacrylamide nanoparticles, prepared by inverse microemulsion polymerization, which contain water-soluble aptamer switch probes (ASPs) trapped in the porous matrix of the nanoparticles. The matrix acts as a molecular fence allowing rapid diffusion of small metabolites into the particles to interact with the aptamer molecules, but at the same time it retains the larger aptamer molecules inside the nanoparticles providing protection against intracellular degradation. We tested the ability of the AptaNPs to measure the adenine-nucleotide content in yeast cells. Our results successfully demonstrate the potential for monitoring any metabolite of interest in living cells by selecting specific aptamers and embedding them in nanoparticles.