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.


Title: Conjugation of Carbon Dots with β-Galactosidase Enzyme: Surface Chemistry and Use in Biosensing
Nanoparticles have been conjugated to biological systems for numerous applications such as self-assembly, sensing, imaging, and therapy. Development of more reliable and robust biosensors that exhibit high response rate, increased detection limit, and enhanced useful lifetime is in high demand. We have developed a sensing platform by the conjugation of β-galactosidase, a crucial enzyme, with lab-synthesized gel-like carbon dots (CDs) which have high luminescence, photostability, and easy surface functionalization. We found that the conjugated enzyme exhibited higher stability towards temperature and pH changes in comparison to the native enzyme. This enriched property of the enzyme was distinctly used to develop a stable, reliable, robust biosensor. The detection limit of the biosensor was found to be 2.9 × 10−4 M, whereas its sensitivity was 0.81 µA·mmol−1·cm−2. Further, we used the Langmuir monolayer technique to understand the surface properties of the conjugated enzyme. It was found that the conjugate was highly stable at the air/subphase interface which additionally reinforces the suitability of the use of the conjugated enzyme for the biosensing applications.  more » « less
Award ID(s):
1809060
PAR ID:
10157864
Author(s) / Creator(s):
; ; ; ; ; ;
Date Published:
Journal Name:
Molecules
Volume:
24
Issue:
18
ISSN:
1420-3049
Page Range / eLocation ID:
3275
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. A chip-based electrochemical biosensor is developed herein for the detection of organophosphate (OP) in food materials. The principle of the sensing platform is based on the inhibition of dimethoate (DMT), a typical OP that specifically inhibits acetylcholinesterase (AChE) activity. Carbon nanotube-modified gold electrodes functionalized with polydiallyldimethylammonium chloride (PDDA) and oxidized nanocellulose (NC) were investigated for the sensing of OP, yielding high sensitivity. Compared with noncovalent adsorption and deposition in bovine serum albumin, bioconjugation with lysine side chain activation allowed the enzyme to be stable over three weeks at room temperature. The total amount of AChE was quantified, whose activity inhibition was highly linear with respect to DMT concentration. Increased incubation times and/or DMT concentration decreased current flow. The composite electrode showed a sensitivity 4.8-times higher than that of the bare gold electrode. The biosensor was challenged with organophosphate-spiked food samples and showed a limit of detection (LOD) of DMT at 4.1 nM, with a limit of quantification (LOQ) at 12.6 nM, in the linear range of 10 nM to 1000 nM. Such performance infers significant potential for the use of this system in the detection of organophosphates in real samples. 
    more » « less
  2. Oxidative stress and excessive accumulation of the superoxide (O2.-) anion are at the genesis of many pathological conditions and the onset of several diseases. The real time monitoring of (O2.-) release is important to assess the extent of oxidative stress in these conditions. Herein, we present the design, fabrication and characterization of a robust (O2.-) biosensor using a simple and straightforward procedure involving deposition of a uniform layer of L-Cysteine on a gold wire electrode to which Cytochrome C (Cyt c) was conjugated. The immobilized layers, studied using conductive Atomic Force Microscopy (c-AFM) revealed a stable and uniformly distributed redox protein on the gold surface, visualized as conductivity and surface topographical plots. The biosensor enabled detection of (O2.-) at an applied potential of 0.15 V with a sensitivity of 42.4 nA/μM and a detection limit of 2.4 nM. Utility of the biosensor was demonstrated in measurements of real time (O2.-) release in activated human blood platelets and skeletal rat limb muscles following ischemia reperfusion injury (IRI), confirming the biosensor's stability and robustness for measurements in complex biological systems. The results demonstrate the ability of these biosensors to monitor real time release of (O2.-) and estimate the extent of oxidative injury in models that could easily be translated to human pathologies. 
    more » « less
  3. Nanomaterials have been extensively explored in developing sensors due to their unique properties, contributing to the development of reliable sensor designs with improved sensitivity and specificity. Herein, we propose the construction of a fluorescent/electrochemical dual-mode self-powered biosensor for advanced biosensing using DNA-templated silver nanoclusters (AgNCs@DNA). AgNC@DNA, due to its small size, exhibits advantageous characteristics as an optical probe. We investigated the sensing efficacy of AgNCs@DNA as a fluorescent probe for glucose detection. Fluorescence emitted by AgNCs@DNA served as the readout signal as a response to more H2O2 being generated by glucose oxidase for increasing glucose levels. The second readout signal of this dual-mode biosensor was utilized via the electrochemical route, where AgNCs served as charge mediators between the glucose oxidase (GOx) enzyme and carbon working electrode during the oxidation process of glucose catalyzed by GOx. The developed biosensor features low-level limits of detection (LODs), ~23 μM for optical and ~29 μM for electrochemical readout, which are much lower than the typical glucose concentrations found in body fluids, including blood, urine, tears, and sweat. The low LODs, simultaneous utilization of different readout strategies, and self-powered design demonstrated in this study open new prospects for developing next-generation biosensor devices. 
    more » « less
  4. null (Ed.)
    Lab-on-a-chip technology offers an ideal platform for low-cost, reliable, and easy-to-use diagnostics of key biomarkers needed for early screening of diseases and other health concerns. In this work, a graphene field-effect transistor (GFET) functionalized with target-binding aptamers is used as a biosensor for the detection of thrombin protein biomarker. Furthermore, this GFET is integrated with a microfluidic device for enhanced sensing performances in terms of detection limit, sensitivity, and continuous monitoring. Under this platform, a picomolar limit of detection was achieved for measuring thrombin; in our experiment measured as low as 2.6 pM. FTIR, Raman and UV-Vis spectroscopy measurements were performed to confirm the device functionalization steps. Based on the concentration-dependent calibration curve, a dissociation constant of K D = 375.8 pM was obtained. Continuous real-time measurements were also conducted under a constant gate voltage ( V GS ) to observe the transient response of the sensor when analyte was introduced to the device. The target selectivity of the sensor platform was evaluated and confirmed by challenging the GFET biosensor with various concentrations of lysozyme protein. The results suggest that this device technology has the potential to be used as a general diagnostic platform for measuring clinically relevant biomarkers for point-of-care applications. 
    more » « less
  5. Simple and fast detection of small molecules is critical for health and environmental monitoring. Methods for chemical detection often use mass spectrometers or enzymes; the former relies on expensive equipment, and the latter is limited to those that can act as enzyme substrates. Affinity reagents like antibodies can target a variety of small-molecule analytes, but the detection requires the successful design of chemically conjugated targets or analogs for competitive binding assays. Here, we developed a generalizable method for the highly sensitive and specific in-solution detection of small molecules, using cannabidiol (CBD) as an example. Our sensing platform uses gold nanoparticles (AuNPs) functionalized with a pair of chemically induced dimerization (CID) nanobody binders (nanobinders), where CID triggers AuNP aggregation and sedimentation in the presence of CBD. Despite moderate binding affinities of the two nanobinders to CBD (equilibrium dissociation constants KD of ∼6 and ∼56 μM), a scheme consisting of CBD−AuNP preanalytical incubation, centrifugation, and electronic detection (ICED) was devised to demonstrate a high sensitivity (limit of detection of ∼100 picomolar) in urine and saliva, a relatively short sensing time (∼2 h), a large dynamic range (5 logs), and a sufficiently high specificity to differentiate CBD from its analog, tetrahydrocannabinol. The high sensing performance was achieved with the multivalency of AuNP sensing, the ICED scheme that increases analyte concentrations in a small assay volume, and a portable electronic detector. This sensing system is readily applicable for wide molecular diagnostic applications. 
    more » « less