Low Concentration Of Biomarkers Research Articles

Alzheimer's Disease Diagnosis Using Misfolding Proteins in Blood.

Alzheimer's disease (AD) is pathologically characterized by a long progressive phase of neuronal changes, including accumulation of extracellular amyloid-β (Aβ) and intracellular neurofibrillary tangles, before the onset of observable symptoms. Many efforts have been made to develop a blood-based diagnostic method for AD by incorporating Aβ and tau as plasma biomarkers. As blood tests have the advantages of being highly accessible and low cost, clinical implementation of AD blood tests would provide preventative screening to presymptomatic individuals, facilitating early identification of AD patients and, thus, treatment development in clinical research. However, the low concentration of AD biomarkers in the plasma has posed difficulties for accurate detection, hindering the development of a reliable blood test. In this review, we introduce three AD blood test technologies emerging in South Korea, which have distinctive methods of heightening detection sensitivity of specific plasma biomarkers. We discuss in detail the multimer detection system, the self-standard analysis of Aβ biomarkers quantified by interdigitated microelectrodes, and a biomarker ratio analysis comprising Aβ and tau.

Extracellular Vesicles in Saliva as Biomarkers of Exposure and Effect

TPS 651: Air pollution exposure modeling 1, Exhibition Hall, Ground floor, August 27, 2019, 3:00 PM - 4:30 PM Background: Saliva collection is fast, easy, inexpensive, and non-invasive and saliva is easy to transport, making it an ideal biofluid for epidemiological research. However, there is a relatively low concentration of biomarkers in saliva when compared to other biofluids. Thus, successful measurement of salivary analytes such as extracellular vesicle (EV) microRNA requires optimal collection, processing, and storage procedures. Here, we demonstrate the feasibility of collecting multiple saliva samples from participants in field research and show that we can obtain high-quality molecular biomarkers such as EV microRNA. Methods: Subjects refrained from eating, drinking, or oral hygiene for at least one hour prior to collection. Samples were then collected by passive drool. Saliva was kept on ice and processed within one hour of collection. After removing cells and debris, EVs were isolated from cell-free saliva supernatant using an exosome precipitation solution. Re-suspended EVs underwent nanoparticle tracking analysis to assess EV size and concentration. EV total RNA was extracted and RNA quality and yield were assessed using a BioAnalyzer (Agilent). Results: In an initial pilot, we collected five samples from two individuals. Extracted saliva EVs contained up to 90% microRNA. Across participants, saliva EVs had an average concentration of 1.5E+10 particles/mL, with an average size of 363nm (sd: 191nm). Exploratory analyses examining the association between microRNA concentration and personal exposure measurements taken while bicycling outdoors (e.g. particulate matter dose, black carbon dose) suggest that there is a positive association between air pollution exposure while bicycling and saliva EV microRNA concentration. Conclusion: Low volume starting inputs (500uL-1mL) of whole saliva can yield high quality salivary EV microRNA in quantities sufficient for downstream applications such as RNA sequencing. Subsequent analysis of microRNA expression profiles and changes in microRNA levels in saliva may lead to biomarkers of exposure to environmental toxicants.

Controlling the Bioreceptor Spatial Distribution at the Nanoscale for Single Molecule Counting in Microwell Arrays.

The ability to detect low concentrations of protein biomarkers is crucial for the early-stage detection of many diseases and therefore indispensable for improving diagnostic devices for healthcare. Here, we demonstrate that by integrating DNA nanotechnologies like DNA origami and aptamers, we can design innovative biosensing concepts for reproducible and sensitive detection of specific targets. DNA origami structures decorated with aptamers were studied as a novel tool to structure the biosensor surface with nanoscale precision in a digital detection bioassay, enabling control of the density, orientation, and accessibility of the bioreceptor to optimize the interaction between target and aptamer. DNA origami was used to control the spatial distribution of an in-house-generated aptamer on superparamagnetic microparticles, resulting in an origami-linked digital aptamer bioassay to detect the main peanut antigen Ara h1 with 2-fold improved signal-to-noise ratio and 15-fold improved limit of detection compared to a digital bioassay without DNA origami. Moreover, the sensitivity achieved was 4 orders of magnitude higher than commercially available and literature-reported enzyme-linked immunosorbent assay techniques. In conclusion, this novel and innovative approach to engineer biosensing interfaces will be of major interest to scientists and clinicians looking for new molecular insights and ultrasensitive detection of a broad range of targets, and, for the next generation of diagnostics.

Systemic inflammatory impact of periodontitis on cognitive impairment.

The role in dementia of systemic inflammation derived from periodontal disease is not fully elucidated. The objective of our study was to examine the impact of inflammation on the relationship between periodontitis and cognitive impairment. We have designed a case (n=171) and control (n=131) study to determine the periodontal health status, grade of cognitive impairment/dementia and systemic inflammation level, the last being measured by analysis of 29 inflammatory biomarkers using multiplex techniques. At the time of sampling, 11 of the 29 inflammatory biomarkers were associated with cognitive impairment in patients with more severe periodontitis. However, the inflammatory response to severe periodontitis was more reduced (lower biomarker concentrations) in cases (with cognitive impairment or dementia) than in (cognitively healthy) controls, an unexpected finding. Based on these results, we cannot confirm that systemic inflammation derived from periodontal disease plays a relevant role in the aetiology of cognitive impairment.

A hinge-based aligner for fast, large-scale assembly of microfluidic chips.

Microfluidics has shown its vitality in scientific research. But the lack of fast and straightforward approaches for aligning chip and easy-to-control on-chip valve still prevent microfluidic chips from becoming powerful commercial products. This work presents an aligner based on hinge structures, which we call a "hinge aligner", for aligning microfluidic chips. Two flat chip holders are connected by a connecting rod so that the chip holders can rotate relative to each other along the connecting rod, in the way a hinge works. The two chip holders contain pre-designed recesses for placing two chips which can align chips with 20μm resolution. Meanwhile, with this hinge aligner, we can easily implement a fully sealed on-chip valve, which can prevent aqueous liquids from leaking even at 80°C for 30min. The real immunoassay result shows aligned microfluidic chips can detect protein with improved reproducibility in both high and low concentration of biomarkers.

Multi-platform Approach for Microbial Biomarker Identification Using Borrelia burgdorferi as a Model.

The identification of microbial biomarkers is critical for the diagnosis of a disease early during infection. However, the identification of reliable biomarkers is often hampered by a low concentration of microbes or biomarkers within host fluids or tissues. We have outlined a multi-platform strategy to assess microbial biomarkers that can be consistently detected in host samples, using Borrelia burgdorferi, the causative agent of Lyme disease, as an example. Key aspects of the strategy include the selection of a macaque model of human disease, in vivo Microbial Antigen Discovery (InMAD), and proteomic methods that include microbial biomarker enrichment within samples to identify secreted proteins circulating during infection. Using the described strategy, we have identified 6 biomarkers from multiple samples. In addition, the temporal antibody response to select bacterial antigens was mapped. By integrating biomarkers identified from early infection with temporal patterns of expression, the described platform allows for the data driven selection of diagnostic targets.

Biomarkers of Exposure among U.S. Adult Cigar Smokers: Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014).

Given the diverse cigar market and limited data on biomarker patterns by cigar type, we compared biomarkers of nicotine and tobacco toxicants among cigar smokers and other groups. Using Wave 1 urinary biomarker data from 5,604 adults in the Population Assessment of Tobacco and Health (PATH) Study, we compared geometric mean concentrations among cigar-only smokers (all cigars and separately for traditional, cigarillo, and filtered cigars), cigarette-only smokers, dual cigar/cigarette smokers, and never users of tobacco. We calculated geometric mean ratios comparing groups with never users adjusting for sex, age, race/ethnicity, education and creatinine. Some day cigar-only smokers had lower biomarker concentrations than every day cigar-only smokers, but higher than never users. Every day cigar-only smokers (n = 61) had lower TNE-2 (cotinine+trans-3'-hydroxycotinine) compared to every day cigarette-only (n = 2217; P < 0.0001) and dual cigar/cigarette smokers (n = 601; P < 0.0001). Several biomarkers, including NNAL (NNK metabolite) and CYMA (metabolite of acrylonitrile), were comparable in these groups. In exploratory analyses, every day filtered cigar-only (n = 7) smokers had higher biomarker concentrations compared with every day traditional cigar-only smokers (n = 12) and cigarillo-only smokers (n = 24). Every day smokers of each cigar type were similar to exclusive cigarette smokers. For some biomarkers, particularly for every day filtered cigar-only smokers, concentrations were higher. For some biomarkers, every day cigar-only smokers were comparable with every day cigarette-only smokers. Exploratory analyses suggest that biomarkers vary by cigar type with every day filtered cigar-only smokers having the highest concentrations. High exposure to harmful constituents among cigar smokers is a continuing health issue.

Which are the best parameters to obtain proteins and malondialdehyde from a tear sample?

Predictive, preventive and personalized medicine (PPPM) is a current concept in healthcare based on the analysis of biomarkers through non-invasive methods. Biomarkers for inflammation and oxidative stress are especially used for screening. Quantification of tear total protein content is important to identify potential, specific biomarkers, such as malondialdehyde concerning oxidative stress. The Schirmer strip test is an accessible and simple method for tear analysis. However, it is limited by the low concentration of biomarkers in the human tear. In this preliminary study, different procedures were compared for the extraction of tear proteins and malondialdehyde. Schirmer strips were used to obtain tears from healthy subjects. Ionic strength and surfactant agents were assessed, as well as different centrifugation parameters. Finally, several volumes of n-butanol on the process of malondialdehyde extraction were evaluated. The results showed that ionic strength strongly influences the extraction process, although most studies have suggested that surfactant agents are the most relevant factor; the most efficient results were obtained using a 2 M solution of NaCl in phosphate buffered saline. Regarding centrifugation, leaving the Schirmer strip tip left hanging outside the tube cap and using 1000 rpm was the best option, which is a lower centrifugation speed than the usually reported on literature. Moreover, 250 μL was the optimal n-butanol volume for malondialdehyde extraction. The importance of this study relies on the increasing relevance of the biomarkers in the field of PPPM and the need of a standardized method to extract biomarkers from the tears, to optimise its use.

Evaluation of analytical performance of immunoassay methods for cTnI and cTnT: From theory to practice.

Current guidelines worldwide recommend cardiac troponins I (cTnI) and T (cTnT) as the biomarkers of choice for the differential diagnosis of acute coronary syndrome (ACS), and the measurement of the 99th upper reference population limit (URL) value for cardiac troponins, with an imprecision of ≤10 CV%. Measuring the 99th URL of cTnI and cTnT is a challenging analytical task due to low biomarker concentrations present in healthy subjects. Therefore, since the year 2006, several manufacturers have established new generation cTnI and cTnT immunoassays with an improved analytical sensitivity in accordance with the quality specifications described in international guidelines, the more recent of which state that only immunoassays that meet the required quality specifications should be considered "high-sensitivity" methods. For the early diagnosis of ACS, and for the stratification of cardiovascular risk in cardiac patients and the general population, high-sensitivity methods should be employed. It is therefore important for laboratory professionals and clinicians to gain a thorough understanding of the analytical performances of immunoassay methods for cTnI and cTnT, especially at low to normal concentration ranges. The aim of the present study was to analyze critical aspects related to definition, analytical performance, pathophysiological interpretations, and the clinical relevance of high-sensitivity cardiac troponin assays.

Exhaled Breath Condensate: Pilot Study of the Method and Initial Experience in Healthy Subjects.

Analysis of Exhaled breath condensate (EBC) is a re-discovered approach to monitoring the course of the disease and reduce invasive methods of patient investigation. However, the major disadvantage and shortcoming of the EBC is lack of reliable and reproducible standardization of the method. Despite many articles published on EBC, until now there is no clear consensus on whether the analysis of EBC can provide a clue to diagnosis of the diseases. The purpose of this paper is to investigate our own method, to search for possible standardization and to obtain our own initial experience. Thirty healthy volunteers provided the EBC, in which we monitored the density, pH, protein, chloride and urea concentration. Our results show that EBC pH is influenced by smoking, and urea concentrations are affected by the gender of subjects. Age of subjects does not play a role. The smallest coefficient of variation between individual volunteers is for density determination. Current limitations of EBC measurements are the low concentration of many biomarkers. Standardization needs to be specific for each individual biomarker, with focusing on optimal condensate collection. EBC analysis has a potential become diagnostic test, not only for lung diseases.

Ichnofabrics and chemostratigraphy argue against persistent anoxia during the Upper Kellwasser Event in New York State

Organic-rich strata coeval with bioevents of varying magnitudes characterize global Devonian sedimentary successions. The Upper Kellwasser (UKw) black shale depositional event is coincident with the largest pulse of diversity loss within the marine Late Devonian mass extinction and has been shown to be an ecologically critical turnover for shallow-water species. Marine anoxia/euxinia is widely thought to be an important and ubiquitous driver of this biotic crisis, though the duration, intensity, and global extent of these environmental conditions during the UKw event are not well-constrained.We characterized redox conditions during deposition of the UKw in the northern Appalachian Basin to constrain local variability and relative magnitude of dissolved oxygen fluctuations. We used a combination of proxies relating to the bottom waters, water column, and photic zone to compile an integrated picture of basinal oxygen dynamics. Our multi-faceted approach combines trace fossil evidence for faunal activity at the sediment-water interface with inorganic and organic geochemical proxies for redox conditions within the water column. Minor biological disruptions to laminated sedimentary fabric indicate at least intermittent oxygenation of the bottom waters during deposition of the UKw, albeit in a low dissolved oxygen seafloor setting. Trace metal proxies (Mo, Mn, U and V) and Fe mineral speciation provide compelling evidence for a water column experiencing intermittent rather than persistent anoxic/euxinic conditions. Lipid biomarkers reinforce the interpretation of variable dissolved oxygen conditions with very low concentrations of Chlorobi carotenoid biomarkers pointing to seasonal/rare episodes of photic zone euxinia. This integrated dataset provides evidence for local redox variability in UKw deposition and supports the likelihood that marine anoxia and euxinia were not globally persistent at the Frasnian-Famennian boundary.

Urinary concentrations of 3-(diethylcarbamoyl)benzoic acid (DCBA), a major metabolite of N,N-diethyl-m-toluamide (DEET) and semen parameters among men attending a fertility center.

Are specific gravity (SG)-adjusted urinary concentrations of 3-(diethylcarbamoyl)benzoic acid (DCBA) associated with semen parameters among men attending an academic fertility center? Our study did not demonstrate any association between SG-adjusted urinary DCBA concentrations and semen parameters among men attending an academic fertility center. N,N-Diethyl-m-toluamide (DEET) is the most common active ingredient in consumer insect repellents. The recent rise in public health concerns regarding mosquito-borne diseases such as Zika, have led to an increased use of DEET insect repellents, especially among couples planning pregnancy. Animal studies have observed reproductive toxicity from DEET exposure. However, the reproductive health effects of DEET and its metabolites on human reproduction are unknown. Between 2007 and 2015, 90 men participating in a prospective cohort study at the Massachusetts General Hospital Fertility Center provided 171 urine samples and 250 semen samples for analysis. The urinary concentrations of DEET, N,N-diethyl-3-hydroxymethylbenzamide (DHMB) and DCBA were quantified by isotope-dilution tandem mass spectrometry and adjusted by SG. We used linear mixed models to evaluate the association between tertiles of SG-adjusted urinary DCBA concentrations and semen parameters (semen volume, sperm concentration, total sperm count, progressive motility, total progressive motility count, normal morphology and total normal morphology count), adjusting for covariates. DEET and DHMB were not considered for analysis because of the low percentage of detectable concentrations (<7%). Effect modification by BMI and smoking status was explored. Participants had a median age of 36 years and BMI of 27 kg/m2, and 68% had never smoked. The SG-adjusted geometric mean DCBA urinary concentration was 2.20 μg/l, with 85% detection frequency. The majority of semen parameters fell within the normal range with the exception of progressive motility, where 64% of the men had values below the WHO 2010 lower reference limits. SG-adjusted urinary DCBA concentrations were not associated with semen parameters in unadjusted or adjusted models. Men in the highest tertile of SG-adjusted urinary DCBA concentrations had comparable semen parameters to men in the lowest tertile (2.59 vs. 2.88 ml for semen volume, 47.9 vs. 45.8 million/ml for sperm concentration, 116 vs. 118 million for total sperm count, 25 vs. 24% for progressive sperm motility, and 6.1 vs. 5.8% for morphologically normal sperm). In addition, BMI and smoking status did not modify the associations. We had a relatively small sample size with similar socioeconomic backgrounds and with overall relatively low urinary concentrations of DEET biomarkers. However, our sample size was enough to detect moderate differences with at least 80% statistical power, between the first and third tertiles of urinary DCBA concentrations. Limitations also include possible misclassification of DCBA exposure and difficulties in extrapolating the findings to the general population. Our study found no associations between urinary concentrations of DCBA, a major metabolite of the insect repellent DEET, and semen parameters in men presenting for infertility treatment. While these results are reassuring, further studies including larger sample sizes and higher exposures are warranted. The project was financed by the National Institute of Health grants R01ES022955 and R01ES009718 and by grant P30ES000002 from the National Institute of Environmental Health Sciences (NIEHS). None of the authors has any conflicts of interest to declare. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. N/A.

Theranostic Contact Lens for Modulation and Detection of Viral Infection Richard Newell

Ocular fluid is an extracellular fluid excreted from the tear gland. Several important markers from ocular fluid have been identified as having significant clinical diagnostic value for various diseases. The contact lens is disposable, relatively cheap and serves as a platform to obtain direct intimate contact with ocular fluid and is therefore an attractive and a promising platform for point-of-care diagnostic tests. Here, we present an entirely new concept of a wearable theranostic device, in the form of a contact lens (theranostic lens) with a dual-function hybrid surface, to modulate and detect a pathogenic attack by herpes simplex virus type 1 (HSV-1). The theranostic lenses were constructed using a Layer-by-Layer (LbL) surface engineering technique to produce a functional hybrid surface containing both an anti-HSV-1 polysulfonate compound and specific antibodies for viral detection. The resulting theranostic lenses retained good optically transparency and surface wettability, and were non-toxic towards human corneal epithelial cells (HCECs). We showed that the theranostic lenses could capture and concentrate interleukin-1α (IL-1α), which is upregulated during HSV-1 infection, using an artificial cornea model integrated with a microfluidic system mimicking tear flow. The theranostics lenses exhibited effective anti-HSV-1 activity and good analytical performance for the detection of IL-1α, with a limit of detection of 1.43 pg mL-1 and a wide linear range covering the clinically relevant region. Our strategy also tackles the major problems in tear diagnostics that are associated with the small volumes from tear sampling and the low concentration of biomarkers in these samples. This work offers a new paradigm for wearable, non-invasive healthcare devices combining diagnosis and protection against disease. We believe that this approach holds promise as a next-generation, point-of-care and de-centralized diagnostic/theranostic platform for a range of biomarkers.

An ultrasensitive enzyme-free electrochemical immunosensor based on redox cycling amplification using methylene blue.

We report a new enzyme-free electrochemical sensor for ultrasensitive measurements of protein biomarkers in plasma and whole blood samples based on a unique electrochemical-chemical-chemical (ECC) redox cycling signal amplification scheme. This scheme uses methylene blue (MB) as a redox indicator which undergoes an endergonic reaction with Ru(NH3)63+ and a highly exergonic reaction with tris(2-carboxyethyl)phosphine (TCEP). This approach offers improved detection sensitivity and sensor stability compared with enzyme-based ECC redox cycling techniques, while involving a simpler sensor modification process and detection protocol. This redox cycling scheme was combined with a robust immunosandwich assay for quantitative measurements of protein biomarkers. For proof of principle, Plasmodium falciparum histidine-rich protein 2 (PfHRP2) was measured in human plasma and whole blood samples, which could be detected down to 10 fg mL-1 and 18 fg mL-1, respectively. Furthermore, this immunosensor exhibits high selectivity, excellent reproducibility and good stability for up to 2 weeks, making it a promising platform for point-of-care testing, especially for detecting extremely low biomarker concentrations in raw biofluids.

Redistribution of soil organic matter by permafrost disturbance in the Canadian High Arctic

With increased warming in the Arctic, permafrost thaw may induce localized physical disturbance of slopes. These disturbances, referred to as active layer detachments (ALDs), redistribute soil across the landscape, potentially releasing previously unavailable carbon (C). In 2007–2008, widespread ALD activity was reported at the Cape Bounty Arctic Watershed Observatory in Nunavut, Canada. Our study investigated organic matter (OM) composition in soil profiles from ALD-impacted and undisturbed areas. Solid-state 13C nuclear magnetic resonance (NMR) and solvent-extractable biomarkers were used to characterize soil OM. Throughout the disturbed upslope profile, where surface soils and vegetation had been removed, NMR revealed low O-alkyl C content and biomarker analysis revealed low concentrations of solvent-extractable compounds suggesting enhanced erosion of labile-rich OM by the ALD. In the disturbed downslope region, vegetation remained intact but displaced material from upslope produced lateral compression ridges at the surface. High O-alkyl content in the surface horizon was consistent with enrichment of carbohydrates and peptides, but low concentrations of labile biomarkers (i.e., sugars) suggested the presence of relatively unaltered labile-rich OM. Decreased O-alkyl content and biomarker concentrations below the surface contrasted with the undisturbed profile and may indicate the loss of well-established pre-ALD surface drainage with compression ridge formation. However, pre-ALD profile composition remains unknown and the observed decreases may result from nominal pre-ALD OM inputs. These results are the first to establish OM composition in ALD-impacted soil profiles, suggesting reallocation of permafrost-derived soil C to areas where degradation or erosion may contribute to increased C losses from disturbed Arctic soils.

A deglaciation and Holocene biomarker-based reconstruction of climate and environmental variability in NW Iberian Peninsula: the Sanabria Lake sequence

The molecular biomarker composition of two sediment cores from Sanabria Lake (NW Iberian Peninsula) and a survey of modern plants in the watershed provide a reconstruction of past vegetation and landscape dynamics since deglaciation. During a proglacial stage in Lake Sanabria (prior to 14.7 cal ka BP), very low biomarker concentration and carbon preference index (CPI) values ~1 suggest that the n-alkanes could have derived from eroded ancient sediment sources or older organic matter with high degree of maturity. During the Late glacial (14.7–11.7 cal ka BP) and the Holocene (last 11.7 cal ka BP) intervals with higher biomarker and triterpenoid concentrations (high %nC29 , nC31 alkanes), higher CPI and average carbon length (ACL), and lower Paq (proportion of aquatic plants) are indicative of major contribution of vascular land plants from a more forested watershed (e.g. Mid Holocene period 7.0–4.0 cal ka BP). Lower biomarker concentrations (high %nC27 alkanes), CPI and ACL values responded to short phases with decreased allochthonous contribution into the lake that correspond to centennial-scale periods of regional forest decline (e.g. 4–3 ka BP, Roman deforestation after 2.0 ka, and some phases of the LIA, seventeenth–nineteenth centuries). Human activities in the watershed were significant during early medieval times (1.3–1.0 cal ka BP) and since 1960 CE, in both cases associated with relatively higher productivity stages in the lake (lower biomarker and triterpenoid concentrations, high %nC23 and %nC31 respectively, lower ACL and CPI values and higher Paq). The lipid composition of Sanabria Lake sediments indicates a major allochthonous (watershed-derived) contribution to the organic matter budget since deglaciation, and a dominant oligotrophic status during the lake history. The study constrains the climate and anthropogenic forcings and watershed versus lake sources in organic matter accumulation processes and helps to design conservation and management policies in mountain, oligotrophic lakes.

Life Sciences Discovery and Technology Highlights

Life Sciences Discovery and Technology Highlights

Integration of an optical CMOS sensor with a microfluidic channel allows a sensitive readout for biological assays in point-of-care tests

In this manuscript, a microfluidic detection module, which allows a sensitive readout of biological assays in point-of-care (POC) tests, is presented. The proposed detection module consists of a microfluidic flow cell with an integrated Complementary Metal–Oxide–Semiconductor (CMOS)-based single photon counting optical sensor. Due to the integrated sensor-based readout, the detection module could be implemented as the core technology in stand-alone POC tests, for use in mobile or rural settings. The performance of the detection module was demonstrated in three assays: a peptide, a protein and an antibody detection assay. The antibody detection assay with readout in the detection module proved to be 7-fold more sensitive that the traditional colorimetric plate-based ELISA. The protein and peptide assay showed a lower limit of detection (LLOD) of 200fM and 460fM respectively. Results demonstrate that the sensitivity of the immunoassays is comparable with lab-based immunoassays and at least equal or better than current mainstream POC devices. This sensitive readout holds the potential to develop POC tests, which are able to detect low concentrations of biomarkers. This will broaden the diagnostic capabilities at the clinician’s office and at patient’s home, where currently only the less sensitive lateral flow and dipstick POC tests are implemented.

Surface-Engineered Contact Lens as an Advanced Theranostic Platform for Modulation and Detection of Viral Infection.

We have demonstrated an entirely new concept of a wearable theranostic device in the form of a contact lens (theranostic lens) with a dual-functional hybrid surface to modulate and detect a pathogenic attack, using a the corneal HSV serotype-1 (HSV-1) model. The theranostic lenses were constructed using a facile layer-by-layer surface engineering technique, keeping the theranostic lenses with good surface wettability, optically transparency, and nontoxic toward human corneal epithelial cells. The theranostic lenses were used to capture and concentrate inflammatory cytokines such as interleukin-1α (IL-1α), which is upregulated during HSV-1 reactivation, for sensitive, noninvasive diagnostics. The theranostic lens also incorporated an antiviral coating to serve as a first line of defense to protect patients against disease. Our strategy tackles major problems in tear diagnostics that are mainly associated with the sampling of a relatively small volume of fluid and the low concentration of biomarkers. The theranostic lenses show effective anti-HSV-1 activity and good analytical performance for the detection of IL-1α, with a limit of detection of 1.43 pg mL(-1) and a wide linear range covering the clinically relevant region. This work offers a new paradigm for "wearable" noninvasive healthcare devices combining "diagnosis" and "protection" against disease, while supporting patient compliance. We believe that this approach holds immense promise as a next-generation point-of-care and decentralized diagnostic/theranostic platform for a range of biomarkers.

Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism.