skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Multiplexing cytokine analysis: towards reducing sample volume needs in clinical diagnostics
The trend for improved more precise diagnostics and management of disease heavily relies on the measurement of panels of biomarkers in physiological samples of patients. Ideally, the ultimate goal would be to detect as many clinically relevant biomarkers as possible in a single drop of blood, achieving quick, sensitive, reproducible, and affordable detection in small volume physiological samples. Bioluminescent (BL) proteins provide many of the desired characteristics required for such labels, including detection at extremely low concentrations, no interference from physiological fluids leading to excellent detection limits, and compatibility with many miniaturized systems. However, to date the use of BL proteins has been restricted by their limited multiplexing capabilities. BL proteins typically exhibit a single emission profile and decay kinetics making the simultaneous detection of multiple analytes difficult. Recent progresses in this area include the use of two different engineered luminescent proteins to achieve resolved signals via one-dimensional time resolution. This approach, however, to date only lead to a dual analyte detection. Herein, we have demonstrated that using a two-dimensional approach that combines both temporal and spatial resolution, we can expand the multiplexing capabilities of bioluminescent proteins. To that end, the photoprotein aequorin (AEQ) has been employed for the simultaneous detection of three separate analytes in a single well, differentiated through the use of three discrete time/wavelength windows. Through a combination of site-specific mutations and synthetic coelenterazines “semi-synthetic” AEQ variants have been developed with altered emission profiles and decay kinetics. In this study, two AEQ mutant proteins were genetically conjugated to three pro-inflammatory cytokines (tumor necrosis factor alpha, interleukins 6 and 8) resulting in AEQ-labeled cytokines. These fusion proteins were combined with synthetic coelenterazines resulting in proteins with differing emission maxima and half-lives to allow for the simultaneous detection of all three cytokines in a single sample. The validity of the assay was demonstrated in serum by employing human physiological samples and comparing our results with commercially available individual tests for each of the three cytokines.  more » « less
Award ID(s):
1841419
PAR ID:
10125906
Author(s) / Creator(s):
; ; ; ; ;
Date Published:
Journal Name:
The Analyst
Volume:
144
Issue:
10
ISSN:
0003-2654
Page Range / eLocation ID:
3250 to 3259
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ; ; ; et al (, Neurophotonics)
    Significance Bioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). Aim To further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation. Approach We developed novel luciferases optimized for Förster resonance energy transfer when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). Results A new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard) and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. Conclusions We report a robust new option for achieving multiple modes of control in a single actuator and a promising engineering strategy for continued improvement of BL-OG. 
    more » « less
  2. ; ; ; ; (, Journal of Neuroscience Research)
    Abstract BioLuminescent (BL) light production can modulate neural activity and behavior through co‐expressed OptoGenetic (OG) elements, an approach termed “BL‐OG.” Yet, the relationship between BL‐OG effects and bioluminescent photon emission has not been characterizedin vivo. Further, the degree to which BL‐OG effects strictly depend on optogenetic mechanisms driven by bioluminescent photons is unknown. Crucial to every neuromodulation method is whether the activator shows a dynamic concentration range driving robust, selective, and nontoxic effects. We systematically tested the effects of four key components of the BL‐OG mechanism (luciferin, oxidized luciferin, luciferin vehicle, and bioluminescence), and compared these against effects induced by the Luminopsin‐3 (LMO3) BL‐OG molecule, a fusion of slow burn Gaussia luciferase (sbGLuc) and Volvox ChannelRhodopsin‐1 (VChR1). We performed combined bioluminescence imaging and electrophysiological recordings while injecting specific doses of Coelenterazine (substrate for sbGluc), Coelenteramide (CTM, the oxidized product of CTZ), or CTZ vehicle. CTZ robustly drove activity in mice expressing LMO3, with photon production proportional to firing rate. In contrast, low and moderate doses of CTZ, CTM, or vehicle did not modulate activity in mice that did not express LMO3. We also failed to find bioluminescence effects on neural activity in mice expressing an optogenetically nonsensitive LMO3 variant. We observed weak responses to the highest dose of CTZ in control mice, but these effects were significantly smaller than those observed in the LMO3 group. These results show that in neocortexin vivo, there is a large CTZ range wherein BL‐OG effects are specific to its active chemogenetic mechanism. 
    more » « less
  3. ; ; ; ; ; ; ; (, Scientific Reports)
    Abstract Proteins are useful biomarkers for a wide range of applications such as cancer detection, discovery of vaccines, and determining exposure to viruses and pathogens. Here, we present a low-noise front-end analog circuit interface towards development of a portable readout system for the label-free sensing of proteins using Nanowell array impedance sensing with a form factor of approximately 35 cm 2 . The electronic interface consists of a low-noise lock-in amplifier enabling reliable detection of changes in impedance as low as 0.1% and thus detection of proteins down to the picoMolar level. The sensitivity of our system is comparable to that of a commercial bench-top impedance spectroscope when using the same sensors. The aim of this work is to demonstrate the potential of using impedance sensing as a portable, low-cost, and reliable method of detecting proteins, thus inching us closer to a Point-of-Care (POC) personalized health monitoring system. We have demonstrated the utility of our system to detect antibodies at various concentrations and protein (45 pM IL-6) in PBS, however, our system has the capability to be used for assaying various biomarkers including proteins, cytokines, virus molecules and antibodies in a portable setting. 
    more » « less
  4. ; ; ; ; ; ; ; ; ; (, Neurophotonics)
    Significance Pain comprises a complex interaction between motor action and somatosensation that is dependent on dynamic interactions between the brain and spinal cord. This makes understanding pain particularly challenging as it involves rich interactions between many circuits (e.g., neural and vascular) and signaling cascades throughout the body. As such, experimentation on a single region may lead to an incomplete and potentially incorrect understanding of crucial underlying mechanisms. Aim We aimed to develop and validate tools to enable detailed and extended observation of neural and vascular activity in the brain and spinal cord. The first key set of innovations was targeted to developing novel imaging hardware that addresses the many challenges of multisite imaging. The second key set of innovations was targeted to enabling bioluminescent (BL) imaging, as this approach can address limitations of fluorescent microscopy including photobleaching, phototoxicity, and decreased resolution due to scattering of excitation signals. Approach We designed 3D-printed brain and spinal cord implants to enable effective surgical implantations and optical access with wearable miniscopes or an open window (e.g., for one- or two-photon microscopy or optogenetic stimulation). We also tested the viability for BL imaging and developed a novel modified miniscope optimized for these signals (BLmini). Results We describe “universal” implants for acute and chronic simultaneous brain–spinal cord imaging and optical stimulation. We further describe successful imaging of BL signals in both foci and a new miniscope, the “BLmini,” which has reduced weight, cost, and form-factor relative to standard wearable miniscopes. Conclusions The combination of 3D-printed implants, advanced imaging tools, and bioluminescence imaging techniques offers a coalition of methods for understanding spinal cord–brain interactions. Our work has the potential for use in future research into neuropathic pain and other sensory disorders and motor behavior. 
    more » « less
  5. ; ; ; ; ; (, Communications Chemistry)
    Abstract Single-molecule fluorescence experiments have transformed our understanding of complex materials and biological systems. Whether single molecules are used to report on their nano-environment or provide for localization, understanding their blinking dynamics (i.e., stochastic fluctuations in emission intensity under continuous illumination) is paramount. We recently demonstrated another use for blinking dynamics called blink-based multiplexing (BBM), where individual emitters are classified using a single excitation laser based on blinking dynamics, rather than color. This study elucidates the structure-activity relationships governing BBM performance in a series of model rhodamine, BODIPY, and anthraquinone fluorophores that undergo different photo-physical and-chemical processes during blinking. Change point detection and multinomial logistic regression analyses show that BBM can leverage spectral fluctuations, electron and proton transfer kinetics, as well as photostability for molecular classification—even within the context of a shared blinking mechanism. In doing so, we demonstrate two- and three-color BBM with ≥ 93% accuracy using spectrally-overlapped fluorophores. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email