The sweet detection of rolling circle amplification : Glucose-based electrochemical genosensor for the detection of viral nucleic acid

• Herein, an isothermal padlock probe-based assay for the simple and portable detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical readout is reported. Infectious diseases remain a constant threat on a global scale, as in recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralising healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. The design of the demonstrated assay consists of a rolling circle amplification (RCA) technique, responsible for the generation of nucleic acid amplicons as RCA products (RCPs). The RCPs are generated on magnetic beads (MB) and subsequently, connected via streptavidin-biotin bonds to GOx. The enzymatic catalysis of glucose by the bound GOx allows for an indirect electrochemical measurement of the DNA target. The RCPs generated on the surface of the MB were confirmed by scanning electron microscopy, and among other experimental conditions such as the type of buffer, temperature, concentration of GOx, sampling and measurement time were evaluated for the optimum electrochemical detection. Accordingly, 125 μg mL−1 of GOx with 5 mM glucose using phosphate buffer saline (PBS), monitored for 1 min were selected as the ideal conditions. Finally, we assessed the analytical performance of the biosensing strategy by using clinical samples of Ebola virus from patients. Overall, this work provides a proof-of-concept bioassay for simple and portable molecular diagnostics of emerging pathogens using electrochemical detection, especially in resource-limited settings.

Ämnesord

NATURVETENSKAP  -- Kemi -- Analytisk kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences -- Analytical Chemistry (hsv//eng)

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

NATURVETENSKAP  -- Kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

Nyckelord

Chronoamperometric readout

Ebola virus

Glucose oxidase

Molecular diagnostics

Point-of-care

Rolling-circle-amplification (RCA)

Diagnosis

Diseases

Glucose

Glucose sensors

Nucleic acids

Product design

Scanning electron microscopy

Viruses

Point of care

Rolling circle amplifications

Chemical detection

biotin

buffer

phosphate buffered saline

streptavidin

virus DNA

Africa

amplicon

analytical parameters

Article

bioassay

biological monitoring

catalysis

concentration (parameter)

controlled study

Ebola hemorrhagic fever

Ebolavirus

electrochemical detection

enzyme binding

epidemic

gene targeting

isotherm

magnetism

measurement

molecular probe

nonhuman

nucleic acid amplification

pandemic

point of care testing

rolling circle amplification

sampling

surface property

temperature sensitivity

Publikations- och innehållstyp

ref (ämneskategori)

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