Efforts toward the continuous monitoring of molecular markers of performance.

OBJECTIVES: Technologies supporting the continuous, real-time measurement of blood oxygen saturation and plasma glucose levels have improved our ability to monitor performance status. Our ability to monitor other molecular markers of performance, however, including the hormones known to indicate overtraining and general health, has lagged. That is, although a number of other molecular markers of performance status have been identified, we have struggled to develop viable technologies supporting their real-time monitoring in the body. Here we review biosensor approaches that may support such measurements, as well as the molecules potentially of greatest interest to monitor. DESIGN: Narrative literature review. METHOD: Literature review. RESULTS: Significant effort has been made to harness the specificity, affinity, and generalizability of biomolecular recognition in a platform technology supporting continuous in vivo molecular measurements. Most biosensor approaches, however, are either not generalizable to most targets, or fail when challenged in the complex environments found in vivo. Electrochemical aptamer-based sensors, in contrast, are the first technology to simultaneously achieve both of these critical attributes. In an effort to illustrate the potential of this platform technology, we both critically review the literature describing it and briefly survey some of the molecular performance markers we believe will prove advantageous to monitor using it. CONCLUSIONS: Electrochemical aptamer-based sensors may be the first truly generalizable technology for monitoring specific molecules in situ in the body and how adaptation of the platform to subcutaneous microneedles will enable the real-time monitoring of performance markers via a wearable, minimally invasive device.

Quantification of Opioids in Urine Using an Aptamer-Based Free-Solution Assay.

The opioid epidemic continues in the United States. Many have been impacted by this epidemic, including neonates who exhibit Neonatal Abstinence Syndrome (NAS). Opioid diagnosis and NAS can be negatively impacted by limited testing options outside the hospital, due to poor assay performance, false-negatives, rapid drug clearance rates, and difficulty in obtaining enough specimen for testing. Here we report a small volume urine assay for oxycodone, hydrocodone, fentanyl, noroxycodone, norhydrocodone, and norfentanyl with excellent LODs and LOQs. The free-solution assay (FSA), coupled with high affinity DNA aptamer probes and a compensated interferometric reader (CIR), represents a potential solution for quantifying opioids rapidly, at high sensitivity, and noninvasively on small sample volumes. The mix-and-read test is 5- to 275-fold and 50- to 1250-fold more sensitive than LC-MS/MS and immunoassays, respectively. Using FSA, oxycodone, hydrocodone, fentanyl, and their urinary metabolites were quantified using 10 µL of urine at 28-81 pg/mL, with >95% specificity and excellent accuracy in ∼1 h. The assay sensitivity, small sample size requirement, and speed could enable opioid screening, particularly for neonates, and points to the potential for pharmacokinetic tracking.

Rapid quantification of two chemical nerve agent metabolites in serum.

Organophosphorus compounds (OPs) continue to represent a significant chemical threat to humans due to exposures from their use as weapons, their potential storage hazards, and from their continued use agriculturally. Existing methods for detection include ELISA and mass spectrometry. The new approach presented here provides an innovative first step toward a portable OP quantification method that surmounts conventional limitations involving sensitivity, selectivity, complexity, and portability. DNA affinity probes, or aptamers, represent an emerging technology that, when combined with a mix-and-read, free-solution assay (FSA) and a compensated interferometer (CI) can provide a novel alternative to existing OP nerve agent (OPNA) quantification methods. Here it is shown that FSA can be used to rapidly screen prospective aptamers in the biological matrix of interest, allowing the identification of a 'best-in-class' probe. It is also shown that combining aptamers with FSA-CI enables quantification of the OPNA metabolites, Sarin (NATO designation "G-series, B", or GB) and Venomous Agent X (VX) acids, rapidly with high selectivity at detection limits of sub-10 pg/mL in 25% serum (by volume in PBS). These results suggest there is potential to directly impact diagnostic specificity and sensitivity of emergency response testing methods by both simplifying sample preparation procedures and making a benchtop reader available for OPNA metabolite quantification.

Aptamer-gold nanoparticle conjugates for the colorimetric detection of arboviruses and vector mosquito species.

The real-time, colorimetric detection of analytes via aptamer-gold nanoparticle technology has proven to be an important, emerging technique within the medical field. Of global health importance, the ability to detect vector mosquito species, such as the Aedes (Ae.) aegypti mosquito, and transmitted arboviruses, such as Zika virus, is paramount to mosquito control and surveillance efforts. Herein, we describe the detection of Ae. aegypti salivary protein for vector identification and the detection of Zika virus to assess mosquito infection status by aptamer-gold nanoparticle conjugates. Key to optimization of these diagnostics were gold nanoparticle capping agents and aptamer degree of labelling (i.e., the amount of aptamers per gold nanoparticle). In the present study, detection was achieved for as little as 10 ng Ae. aegypti salivary protein and 1.0 × 105 PFU live Zika virus. These aptamer-gold nanoparticle conjugate diagnostics could one day prove to be useful as deployable nano-based biosensors that provide easy-to-read optical read outs through a straightforward red-to-blue colour change either within a diagnostic solution or atop a card/membrane-based biosensor.

Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and monitoring the effect of secondary therapeutic agents on lysosomal enzyme activity in drug development for the lysosomal storage disorders and allied diseases.

Tools and techniques to measure mitophagy using fluorescence microscopy.

Mitophagy is a specialized form of autophagy that removes damaged mitochondria, thereby maintaining efficient cellular metabolism and reducing cellular stress caused by aberrant oxidative bursts. Deficits in mitophagy underlie several diseases, and a substantial body of research has elucidated key steps in the pathways that lead to and execute autophagic clearance of mitochondria. Many of these studies employ fluorescence microscopy to visualize mitochondrial morphology, mass, and functional state. Studies in this area also examine colocalization/recruitment of accessory factors, components of the autophagic machinery and signaling molecules to mitochondria. In this review, we provide a brief summary of the current understanding about the processes involved in mitophagy followed by a discussion of probes commonly employed and important considerations of the methodologies to study and analyze mitophagy using fluorescence microscopy. Representative data, where appropriate, are provided to highlight the use of key probes to monitor mitophagy. The review will conclude with a consideration of new possibilities for mitophagy research and a discussion of recently developed technologies for this emerging area of cell biology.

BacMam-mediated gene delivery into multipotent mesenchymal stromal cells.

Baculoviruses have been used over the last several decades for high-level protein production in insect cells. Recently, modified baculovirus containing a mammalian promoter, known as BacMam virus, has been shown to give high transduction efficiencies across several cell types with minimal cytopathic effects. Cell types amenable to BacMam transduction include primary and adult stem cells. The shuttle vectors used in the construction of BacMam viruses can hold gene fragments up to 38 kb in size, and multiple BacMam viruses can be used in a single transduction for the delivery of more than one gene. BacMam technology has been used in the delivery and expression of targeted fluorescent protein cellular markers, small interfering RNAi, and extensively in the development of cell-based assays. BacMam offers an ideal method for the delivery and expression of large genes in hard-to-transfect cells such as primary and adult stem cells. In this chapter, we describe methods of generating high titer stocks of BacMam for transducing MSC and their derivatives.

Fluorescent labeling of proteins in living cells using the FKBP12 (F36V) tag.

Over the past decade live cell imaging has become a key technology to monitor and understand the dynamic behavior of proteins in the physiological context of living cells. The visualization of a protein of interest is most commonly achieved by genetically fusing it to green fluorescent protein (GFP) or one of it variants. Considerable effort has been made to develop alternative methods of protein labeling to overcome the intrinsic limitations of fluorescent proteins. In this report we show the optimization of a live cell labeling technology based on the use of a mutant form of FKBP12 (FKBP12(F36V)) in combination with a synthetic high affinity ligand (SLF') that specifically binds to this mutant. It had been previously shown that the use of a fluorescein-conjugated form of SLF' (5'-fluorescein-SLF') allowed the labeling of proteins genetically fused to FKBP-F36V in living cells. Here we describe the identification of novel fluorescent SLF'dye conjugates that allow specific labeling of FKBP12(F36V) fusion proteins in living cells. To further increase the versatility of this technology we developed a number of technical improvements. We implemented the use of pluronics during the labeling process to facilitate the uptake of the SLF'-dye conjugates and the use suppression dyes to reduce background signal. Furthermore, the time and dose dependency of labeling was investigated in order to determine optimal labeling conditions. Finally, the specificity of the FKBP12(F36V) labeling technology was extensively validated by morphological analysis using a diverse set of FKBP12(F36V) fusions proteins. In addition we show a number of different application examples, such as translocation assays, the generation of biosensors, and multiplex labeling in combination with different labeling technologies, such as FlAsH or GFP. In summary we show that the FKBP12(F36V)/SLF' labeling technology has a broad range of applications and should prove useful for the study of protein function in living cells.

Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids.

As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9.

Use of cellular glucose-6-phosphate dehydrogenase for cell quantitation: applications in cytotoxicity and apoptosis assays.

A fluorescence-based microplate assay was developed to quantify cell death based upon the measurement of glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD is a cytosolic enzyme and leaks from cells when plasma membrane integrity is compromised. In this assay, cell death is measured by correlating the activity of extracellular G6PD to the reduction of resazurin to the fluorescent product, resorufin, via a coupled-enzyme reaction. The coupled-enzyme reaction permits rapid signal amplification from small amounts of G6PD, an advantage over assays based on resazurin alone. This assay is rapid, nontoxic, and amenable to high-throughput screening. The assay has a Z' factor of 0.78.

A fluorescent high-throughput assay for double-stranded DNA: the RediPlate PicoGreen assay.

The fluorescent PicoGreen reagent for detection and quantitation of double-stranded DNA has been adapted for high-throughput screening: the RediPlate PicoGreen double-stranded DNA assay format. In the RediPlate PicoGreen assay format, the PicoGreen reagent is predistributed and co-dried into either 96- or 384-well microplates with the excipient trehalose. The user resuspends the dried reagents upon adding DNA, and measures the resulting fluorescence after a five minute incubation. Replicate fluorescence measurements on nominally identical wells have less than a 5% coefficient of variation. The assay is linear from 5 to 500 ng/ml DNA in a 200 micro l volume. The RediPlate PicoGreen assay format retains the advantages of the original PicoGreen reagent - sensitivity, speed, and specificity - but in a high-throughput format.