Fluorescence-based Biosensor Research Articles

Ratiometric Bioluminescence-Based Zinc Biosensor with Nanomolar Sensitivity

In recent years, we and other workers have developed fluorescence-based sensors and biosensors for Zn2+ and other free metal ions in solution, which have proven valuable as tools for the study of the biology of metal ions. Using the sensors in some applications such as in vivo molecular imaging has been challenging, owing to the difficulty of exciting fluorophores deep in tissues which scatter and absorb exciting light effectively. One approach used by investigators to minimize this problem is the use of bioluminescent labels for in vivo imaging, which perforce require no exciting light. We have employed a bioluminescence resonance energy transfer approach with our carbonic anhydrase-based zinc biosensor to produce a ratiometric bioluminescence-based zinc biosensor.

Compound droplet manipulations on fiber arrays.

Recent works demonstrated that fiber arrays may constitute new means of designing open digital microfluidic systems. Various processes, such as droplet motion, fragmentation, trapping, release, mixing and encapsulation, may be achieved on fiber arrays. However, handling a large number of tiny droplets resulting from the mixing of several liquid components is required for developing microreactors, smart sensors or microemulsifying drugs. Here, we show that the manipulation of tiny droplets onto fiber networks allows for creating compound droplets with a high complexity level. Moreover, this cost-effective and adjustable method may also be implemented with optical fibers in order to develop fluorescence-based biosensor.

Novel biosensors based on optimized glycine oxidase.

Glycine is involved in several physiological functions, e.g. as a neurotransmitter in the central nervous system, and sarcosine has been identified as a differential metabolite greatly enhanced during prostate cancer progression and metastasis. Glycine oxidase from Bacillus subtilis (GO) was engineered with the final aim of producing specific analytical systems to detect these small achiral amino acids. Based on in silico analysis, site-saturation mutagenesis was independently performed at 11 positions: a total of 16 single-point GO variants were analyzed. Significantly improved kinetic parameters were observed on glycine for the A54R, H244K-N-Q-R, Y246W and M261R variants. The introduction of multiple mutations then identified the H244K/M261R variant showing a 5.4-fold increase in maximal activity on glycine. With sarcosine as substrate, a number of single-point variants showed increased maximal activity and/or affinity: the kinetic efficiency was increased 6-fold for the M49L variant. Two GO variants with a high substrate specificity ratio for glycine (versus sarcosine, i.e. H244K GO) or for sarcosine (versus glycine, i.e. M49L GO) combined with high substrate affinity were used to set up a simple fluorescence-based biosensor. This optical sensing assay represents a novel, inexpensive and fast tool to assay glycine or sarcosine concentrations in biological samples (detection limit ≤ 0.5 μm).

Invitro platforms for evaluating liver toxicity.

The liver is a heterogeneous organ with many vital functions, including metabolism of pharmaceutical drugs and is highly susceptible to injury from these substances. The etiology of drug-induced liver disease is still debated although generally regarded as a continuum between an activated immune response and hepatocyte metabolic dysfunction, most often resulting from an intermediate reactive metabolite. This debate stems from the fact that current animal and in vitro models provide limited physiologically relevant information, and their shortcomings have resulted in "silent" hepatotoxic drugs being introduced into clinical trials, garnering huge financial losses for drug companies through withdrawals and late stage clinical failures. As we advance our understanding into the molecular processes leading to liver injury, it is increasingly clear that (a) the pathologic lesion is not only due to liver parenchyma but is also due to the interactions between the hepatocytes and the resident liver immune cells, stellate cells, and endothelial cells; and (b) animal models do not reflect the human cell interactions. Therefore, a predictive human, in vitro model must address the interactions between the major human liver cell types and measure key determinants of injury such as the dosage and metabolism of the drug, the stress response, cholestatic effect, and the immune and fibrotic response. In this mini-review, we first discuss the current state of macro-scale in vitro liver culture systems with examples that have been commercialized. We then introduce the paradigm of microfluidic culture systems that aim to mimic the liver with physiologically relevant dimensions, cellular structure, perfusion, and mass transport by taking advantage of micro and nanofabrication technologies. We review the most prominent liver-on-a-chip platforms in terms of their physiological relevance and drug response. We conclude with a commentary on other critical advances such as the deployment of fluorescence-based biosensors to identify relevant toxicity pathways, as well as computational models to create a predictive tool.

Controlled Reduction of Photobleaching in DNA Origami–Gold Nanoparticle Hybrids

The amount of information obtainable from a fluorescence-based measurement is limited by photobleaching: Irreversible photochemical reactions either render the molecules nonfluorescent or shift their absorption and/or emission spectra outside the working range. Photobleaching is evidenced as a decrease of fluorescence intensity with time, or in the case of single molecule measurements, as an abrupt, single-step interruption of the fluorescence emission that determines the end of the experiment. Reducing photobleaching is central for improving fluorescence (functional) imaging, single molecule tracking, and fluorescence-based biosensors and assays. In this single molecule study, we use DNA self-assembly to produce hybrid nanostructures containing individual fluorophores and gold nanoparticles at a controlled separation distance of 8.5 nm. By changing the nanoparticles' size we are able to systematically increase the mean number of photons emitted by the fluorophores before photobleaching.

Optical immunosensor for quantifying C-telopeptide fragments of type II collagen as an osteoarthritis biomarker in urine

A fluorescence-based biosensor for measuring the quantity of C-telopeptide fragments of type II collagen (CTX-II) was developed as an osteoarthritis biomarker in urine. During osteoarthritis progression, joint components such as cartilage collagen are degraded by collagenase-protease and secreted into the serum as CTX-II. To detect CTX-II in urine (uCTX-II) having structural heterogeneity, conventional sandwich format assay does not provide sufficient accuracy. As an alternative, a competitive immunoassay was developed for the quantification of uCTX-II, which uses antibody-conjugated fluoro-microbeads to generate an optical signal. After preparing the biosensing surface, the uCTX-II sample and antibody-optical probe conjugates were reacted on the PEG4-EKGPDP exposed biosensing surface. The optical probes competed with the uCTX-II in the sample for binding to the immobilized PEG4-EKGPDP. This assay was able to detect uCTX-II concentrations between 200 ng/mmol (corrected value vs. creatinine) and 1,400 ng/mmol, encompassing the clinical detection ranges required for osteoarthritis diagnosis. The competitive immunoassay developed for uCTX-II detection was rapid and exhibited good correlation with conventional ELISA methods. This novel competition assay is a promising tool for the diagnosis and monitoring of osteoarthritis using urine samples.

Microbial fluorescence sensing for human neurotensin receptor type 1 using Gα-engineered yeast cells

Neurotensin receptor type-1 (NTSR1) is a member of the G-protein-coupled receptor (GPCR) family. The natural ligand of NTSR1 is neurotensin (NT), a neuromodulator of the central nervous system. Because NT is also involved in many oncogenic actions, the signaling mediator NTSR1 is a significant molecular target in medicinal and therapeutic fields. In the current study, we constructed a fluorescence-based microbial yeast biosensor that can monitor the activation of human NTSR1 signaling responding to its agonist. To increase the sensitivity of the biosensor, a yeast strain with the green fluorescent protein (GFP) reporter gene was genetically engineered to enhance binding with human NTSR1 expressed on the membrane. Following previous reports, the 5 carboxy-terminal amino acid residues of the guanine nucleotide binding protein α-subunit (Gα) in yeast Gpa1p were substituted with the equivalent human Gαq sequences (Gpa1/Gαq transplant). After optimizing the assay conditions, the Gα-engineered yeast demonstrated significantly improved sensing for NTSR1 signaling. Because detection using a GFP fluorescence reporter considerably simplifies the measurement procedure, this microbial fluorescence sensor holds promise for use in the diagnosis of NTSR1-associated diseases and the development of agonists.

Selective Serial Multi-Antibody Biosensing with TOPAS Microstructured Polymer Optical Fibers

We have developed a fluorescence-based fiber-optical biosensor, which can selectively detect different antibodies in serial at preselected positions inside a single piece of fiber. The fiber is a microstructured polymer optical fiber fabricated from TOPAS cyclic olefin copolymer, which allows for UV activation of localized sensor layers inside the holes of the fiber. Serial fluorescence-based selective sensing of Cy3-labelled α-streptavidin and Cy5-labelled α-CRP antibodies is demonstrated.

Detection of the pesticide by functionalised quantum dots as fluorescence-based biosensor

In this paper, we present the new results of biosensor that is made from the surface–modified quantum dots with acetylcholinesterase enzymes (AChE) for optical detection of the pesticides. The quantum dots (QDs) mentioned in this study are CdTe, CdSe/ZnS and CdSe/ZnSe/ZnS - the thick shell QDs are totally new. The results pointed out that all of the quantum dots are fit for the role of transducers in biosensor. In the biosensor, the QD– streptavidine - AChE is used as the substrate for the detection of pesticide. The pesticides used in this work are Parathion Methyl (PM) and Acetamiprid. The acetylthiocholine (ATCh) is used as an indicator of the activity of the AChE enzyme. Alternatively, the organophosphorus (OP) pesticides are the inhibitor for the AChE enzymes. Therefore, the mixture of the pesticide and ATCh is used for the goal of the specification of pesticide. We can detect pesticides by the change in PL intensity of QDs biosensor, with the content ranges from 0.05 ppb to 10 ppb.

Enhanced-acceptor fluorescence-based single cell ATP biosensor monitors ATP in heterogeneous cancer populations in real time

Current methods to monitor cellular ATP do not provide spatial or temporal localization of ATP in single cells in real time or they display imperfect specificity to ATP. Here, we have developed a single cell, Enhanced Acceptor Fluorescence (EAF)-based ATP biosensor to visualize ATP in real time. This biosensor utilizes a modified mimic of the ε-subunits of the Bacillus subtilis F(0)F(1) synthase and is coupled to the EAF fluorophores pairs, GFP and YFP. The sensor was then used to monitor ATP in a heterogeneous glioblastoma multiform cancer cell population. We anticipate that this innovative technology and our better understanding of the ATP machinery will have substantial influence on future translational studies.

Fabrication of fluorescence-based biosensors from functionalized CdSe and CdTe quantum dots for pesticide detection

This paper presents the results on the fabrication of highly sensitive fluorescence biosensors for pesticide detection. The biosensors are actually constructed from the complex of quantum dots (QDs), acetylcholinesterase (AChE) and acetylthiocholine (ATCh). The biosensor activity is based on the change of luminescence from CdSe and CdTe QDs with pH, while the pH is changed with the hydrolysis rate of ATCh catalyzed by the enzyme AChE, whose activity is specifically inhibited by pesticides. Two kinds of QDs were used to fabricate our biosensors: (i) CdSe QDs synthesized in high-boiling non-polar organic solvent and then functionalized by shelling with two monolayers (2-ML) of ZnSe and eight monolayers (8-ML) of ZnS and finally capped with 3-mercaptopropionic acid (MPA) to become water soluble; and (ii) CdTe QDs synthesized in aqueous phase then shelled with CdS. For normal checks the fabricated biosensors could detect parathion methyl (PM) pesticide at very low contents of ppm with the threshold as low as 0.05 ppm. The dynamic range from 0.05 ppm to 1 ppm for the pesticide detection could be expandable by increasing the AChE amount in the biosensor.

A new mitochondrial pH biosensor for quantitative assessment of pancreatic β-cell function

Mitochondrial pH is a key determinant of mitochondrial energy metabolism. We have developed a new fluorescence-based ratiometric pH biosensor using a chloride-insensitive and hydrogen-sensitive probe for direct, quantitative and bleaching-free measurement in a living cell. Fusing this biosensor with a mitochondrial localization signal (MTpHGV) allowed us to determine mitochondrial pH. This new system was applied to measure mitochondrial pH in pancreatic β-cells, in which mitochondrial function plays a pivotal role in insulin secretion. Rat INS1 cells and mouse MIN6 cells are transfected with MTpHGV stably to monitor mitochondrial pH. While carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment rapidly decreased mitochondrial pH in cultured rat MTpHGV-INS-1 cells, MTpHGV-MIN6 cells showed a rapid increase. These data suggest that MTpHGV probe exist in matrix side in INS-1 cells, but on the outer side of mitochondrial inner membrane in MIN6 cells. Moreover, while MTpHGV-INS-1 cells showed a rapid increase of pH by glucose stimulation, mitochondrial pH decreased quickly by glucose stimulation in all MTpHGV-MIN6 cells examined and recovered smoothly. Perfusion study of glucose load in MTpHGV-MIN6 cells under aminooxyacetate (AOA) or 100μM diazoxide showed that this mitochondrial pH acidification was dependent on nicotinamide adenine dinucleotide (NADH) shuttle, but independent from KATP channel. This new system for measuring mitochondrial pH is sensitive across the range of physiologic conditions and may be a useful tool for evaluating mitochondrial function in living cells.

Optical Detection of the Pesticide by Functionalized Quantum Dots as Fluorescence-Based Biosensor

In this work, the results on using biosensor composed from quantum dots as transducer and acetylcholinesterase enzymes (AChE) to detect pesticides optically are presented. The used quantum dots were CdTe, CdSe/ZnS 10 monolayer (ML) and CdSe/ZnSe2ML/ZnS 8 ML – the brand new thick-shell quantum dots (QD). The study results pointed out that the CdSe/ZnS 10 ML and CdSe/ZnSe 2ML/ZnS 8ML quantum dots best fit for the role of transducers in biosensors. In the biosensor, acetylthiocholine (ATCh) is used as indicator for the AChE enzymes to work, since it is a very powerful hydrolyte with the presence of AChE enzymes. Moreover, the organophosphorus (OP) pesticides are the inhibitors for the AChE enzymes, thus, by the biosensors that we designed, we can detect pesticides by the change in photoluminescence (PL) intensity of QDs, with the detection of OP like parathion methyl is 0.05 ppm, and acetamiprid is 2.5 ppm.

Immobilization of Molecularly Imprinted Polymer Nanoparticles in Electrospun Poly(vinyl alcohol) Nanofibers

We describe the fabrication of polymer nanofibers with entrapped molecularly imprinted polymer (MIP) nanoparticles and study their possible use in a fluorescence-based biosensor application. The MIP was imprinted with the fluorescent amino acid derivative dansyl-L-phenylalanine. Poly(vinyl alcohol) was used as a support for MIP nanoparticles because it is water-soluble and can be spun into very thin fibers. The fibers were characterized by atomic force microscopy and optical microscopy, and fluorescence microscopy was used for the characterization of target binding to the MIP. The fibers show close to 100% recovery upon extraction and rebinding of the target molecule. The selectivity of the system has been demonstrated through competitive binding experiments with nonfluorescent analogues boc-L-phenylalanine and boc-D-phenylalanine.

Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled β-lactamase

Backgroundβ-lactamase conjugated with environment-sensitive fluorescein molecule to residue 166 on the Ω-loop near its catalytic site is a highly effective biosensor for β-lactam antibiotics. Yet the molecular mechanism of such fluorescence-based biosensing is not well understood.ResultsHere we report the crystal structure of a Class A β-lactamase PenP from Bacillus licheniformis 749/C with fluorescein conjugated at residue 166 after E166C mutation, both in apo form (PenP-E166Cf) and in covalent complex form with cefotaxime (PenP-E166Cf-cefotaxime), to illustrate its biosensing mechanism. In the apo structure the fluorescein molecule partially occupies the antibiotic binding site and is highly dynamic. In the PenP-E166Cf-cefatoxime complex structure the binding and subsequent acylation of cefotaxime to PenP displaces fluorescein from its original location to avoid steric clash. Such displacement causes the well-folded Ω-loop to become fully flexible and the conjugated fluorescein molecule to relocate to a more solvent exposed environment, hence enhancing its fluorescence emission. Furthermore, the fully flexible Ω-loop enables the narrow-spectrum PenP enzyme to bind cefotaxime in a mode that resembles the extended-spectrum β-lactamase.ConclusionsOur structural studies indicate the biosensing mechanism of a fluorescein-labelled β-lactamase. Such findings confirm our previous proposal based on molecular modelling and provide useful information for the rational design of β-lactamase-based biosensor to detect the wide spectrum of β-lactam antibiotics. The observation of increased Ω-loop flexibility upon conjugation of fluorophore may have the potential to serve as a screening tool for novel β-lactamase inhibitors that target the Ω-loop and not the active site.

Optimization of antibody-conjugated magnetic nanoparticles for target preconcentration and immunoassays

Biosensors based on antibody recognition have a wide range of monitoring applications that apply to clinical, environmental, homeland security, and food problems. In an effort to improve the limit of detection of the Naval Research Laboratory (NRL) Array Biosensor, magnetic nanoparticles (MNPs) were designed and tested using a fluorescence-based array biosensor. The MNPs were coated with the fluorescently labeled protein, AlexaFluor647–chicken IgG (Alexa647–chick IgG). Antibody-labeled MNPs (Alexa647–chick–MNPs) were used to preconcentrate the target via magnetic separation and as the tracer to demonstrate binding to slides modified with anti-chicken IgG as a capture agent. A full optimization study of the antibody-modified MNPs and their use in the biosensor was performed. This investigation looked at the Alexa647–chick–MNP composition, MNP surface modifications, target preconcentration conditions, and the effect that magnetic extraction has on the Alexa647–chick–MNP binding with the array surface. The results demonstrate the impact of magnetic extraction using the MNPs labeled with fluorescent proteins both for target preconcentration and for subsequent integration into immunoassays performed under flow conditions for enhanced signal generation.

Quantitative Comparison of Protein Surface Coverage on Glass Slides and Silver Island Films in Metal-Enhanced Fluorescence-based Biosensing Applications

The use of Metal-Enhanced Fluorescence (MEF) phenomenon in fluorescence-based bioassays affords for increased sensitivity to be realized by incorporating metal nanoparticles onto planar surfaces. The close-range interactions of metal-fluorophores result in increased fluorescence emission from the bioassays, which in turn affords for the detection of target biomolecules at lower concentrations. Moreover, the use of silver nanoparticles increases the photostability of fluorophores improving the detectability of fluorescence emission under prolonged use of excitation light. Although numerous reports on MEF-based biosensing applications exist, the contribution of protein coverage on Silver Island Films (SIFs) on the increased fluorescence emission was never investigated. This work presents our findings on the quantitative comparison of protein surface coverage on SIFs and blank glass slides. In this regard, identical protein bioassay for a model protein (biotinylated bovine serum albumin, b-BSA) on these surfaces is constructed and the relative extent of protein surface coverage on SIFs and blank glass slides was determined using radio-labeled biomolecules. It was found that the total scintillation counts on SIFs and blank glass slides were similar for BSA concentrations ranging from 1 μM to 1 pM, which implies that increased fluorescence in MEF-based biosensing applications is only due to metal-fluorophore interactions.

Quick identification of apoptosis inducer from Isodon eriocalyx by a drug discovery platform composed of analytical high-speed counter-current chromatography and the fluorescence-based caspase-3 biosensor detection

Analytical high-speed counter-current chromatography (HSCCC), a unique liquid-to-liquid separation technology, has an inherent capability to provide perfect fractionation for tracking active ingredients of medicinal herbs, in a quick, efficient, and high-recovery manner. A high throughput screening (HTS) method which utilizes a novel biosensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique, was newly established and proved to be very sensitive in detecting apoptosis induced by various known anticancer drugs. The first combination of both advanced techniques formed an efficient platform for drug discovery and succeeded in quickly identifying the most potent apoptotic constituent of a Chinese herb namely Isodon eriocalyx. The system of n-hexane/ethyl acetate/methanol/water was used as the separation solvent. The solvent ratio was first set at 3:5:3:5 to check the water-soluble part of the crude extract, and then 1:1:1:1 was used to isolate the target compounds. The active fraction was tracked and purified continuously using HSCCC which was guided by the apoptosis detection at gradually decreased drug concentrations. As a result, the most potent apoptosis inducer in this herb was discovered by analytical HSCCC equipped with a 16 ml mini-coil column, using less than 50 ml diphase solvent, from about 50 mg active fraction. It was identified as eriocalyxin B, a well-known antitumor natural product, by NMR analysis of the HSCCC purified fraction.

Characterization of a novel ultra-low refractive index material for biosensor application

Nanoporous materials can provide significant benefits to the field of biosensors. Their size and porous structure makes them an ideal tool for improving sensor performance. This study characterized a novel ultra-low index of refraction nanoporous organosilicate (NPO) material for use as an optical platform for fluorescence-based optical biosensors. While serving as the low index cladding material, the novel coating based on organosilicate nanoparticles also provides an opportunity for a high surface area coating that can be utilized for immobilizing biological probes. Biological molecules were immobilized onto NPO, which was spin-coated on silicon and glass substrates. The biological molecule was composed of Protein A conjugated to AlexaFluor 546 fluorophore and then immobilized onto the NPO substrate via silanization. Sample analysis consisted of spectrofluorometry, FT-IR spectroscopy, scanning electron microscopy, contact angle measurement and ellipsometry. The results showed the presence of emission peaks at 574nm, indicating that the immobilization of Protein A to the NPO material is possible. When compared to Si and glass substrates not coated with NPO, the results showed a 100× and 10× increase in packing density with the NPO-coated films, respectively. Ellipsometric analysis, FT-IR, contact angle, and SEM imaging of the surface immobilized NPO films suggested that while the surface modifications did induce some damage, it did not incur significant changes to its unique characteristics, i.e., pore structure, wettability and index of refraction. It was concluded that NPO films would be a viable sensor substrate to enhance sensitivity and improve sensor performance.

Nanomaterials in fluorescence-based biosensing

Fluorescence-based detection is the most common method utilized in biosensing because of its high sensitivity, simplicity, and diversity. In the era of nanotechnology, nanomaterials are starting to replace traditional organic dyes as detection labels because they offer superior optical properties, such as brighter fluorescence, wider selections of excitation and emission wavelengths, higher photostability, etc. Their size- or shape-controllable optical characteristics also facilitate the selection of diverse probes for higher assay throughput. Furthermore, the nanostructure can provide a solid support for sensing assays with multiple probe molecules attached to each nanostructure, simplifying assay design and increasing the labeling ratio for higher sensitivity. The current review summarizes the applications of nanomaterials--including quantum dots, metal nanoparticles, and silica nanoparticles--in biosensing using detection techniques such as fluorescence, fluorescence resonance energy transfer (FRET), fluorescence lifetime measurement, and multiphoton microscopy. The advantages nanomaterials bring to the field of biosensing are discussed. The review also points out the importance of analytical separations in the preparation of nanomaterials with fine optical and physical properties for biosensing. In conclusion, nanotechnology provides a great opportunity to analytical chemists to develop better sensing strategies, but also relies on modern analytical techniques to pave its way to practical applications.