Ultraviolet-induced in situ gold nanoparticles for point-of-care testing of infectious diseases in loop-mediated isothermal amplification

Authors : Rajamanickam Sivakumar, Vu Phong Dinh and Nae Yoon Lee

Journal:Lab on a chip

Publish:27 Jan 2021

Presenter:姜雨宸

Outline

• Abstract
• Introduction
• Materials and Methods
• Results
• Conclusion

Abstract

• Ultraviolet-induced in situ gold nanoparticles (AuNPs) coupled with loop-mediated isothermal amplification (LAMP) for POCT
• Coronavirus (COVID-19) 、 Enterococcus faecium (E. faecium spp.)
• Designed a portable device that contained arrays of reagent chambers and detection chambers

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Introduction

• Conventional detection methods are associated with several limitations
• Modern biosensors

Loop-mediated isothermal amplification (LAMP)

• the requirement of a complicated thermal cycling step limits the use of PCR in the detection field
• enables the amplification of specific nucleic acid sequences from medium to long-range template strands, and also produces >50 fold more amplicons than PCR
• Detection Method : colorimetric, immunoassay, fluorescence, electrochemical, and electrophoresis

AuNP-based colorimetric assays

• Good biocompatibility, easy conjugation with biomolecules, large surface area, and particular optical properties including high absorption coefficient, surface plasmon band (SPB), surface enhanced Raman scattering (SERS) and Rayleigh resonance scattering
• labeled AuNP unlabeled AuNPs because they are chemically stable and easy to synthesize and modification of the AuNPs requires additional cost and time 

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Bacterial cultures�

Enterococcus faecium

Luria–Bertani broth(5 mL )

NB

at 37 °C for 16 h

shaking at

200 rpm 

 (1 mL)genomic DNA 

 NanoDrop spectrophotometer:

concentration and quality of gDNA 

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stored at 2–8 °C.

Method�Colorimetric detection of the LAMP amplicons�

• Aqueous solutions of 10 mM HAuCl₄ and 38.8 mM Na₃Ct were added to the LAMP amplicons (4 μL) in the microtube
• Keep under UV light (at 312 nm) for 10 min
• The positive sample that contained a DNA template turned from colorless to red, and the negative sample (no DNA template) remained colorless

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Method�Portable device fabrication�

• Nine detection chambers (4 mm in diameter) and nine reagent chambers (3 mm in diameter) 
• The colorimetric agents and LAMP amplicons were added to the reagent and detection chambers
• The portable device was then kept for amplification at 63 °C for 30 min
• The reagent chamber was folded to make contact with the detection chamber
• The portable device was kept under UV (312 nm, 95 W) for 10 min

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PMMA

(2 mm thickness)

Results and discussion�

Optimization of isothermal conditions for LAMP amplicon detection��

Test the different ranges of incubation time (15–45 min) and temperatures (60–65 °C)

• reaction temperature
• reaction time
• LOD of the LAMP assay

Result:

An incubation temperature of 63 °C for 30 min showed high-color intensity

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Results of agarose gel electrophoresis

Optimization of various volumes and concentrations of HAuCl₄ for LAMP amplicon detection

Various volumes of HAuCl₄ for LAMP amplicon detection

• Various volumes of 10 mM HAuCl4 were then mixed with LAMP amplicons to produce different HAuCl4
• HAuCl₄ : LAMP amplicon volume ratios from 9 : 1 to 4 : 6  
• 38.8 mM Na₃Ct (1 μL) was added to each of the HAuCl₄

Result : the 6 : 4 ratio displayed a dark red color in less than 10 min

Various concentrations of HAuCl₄ for the LAMP amplicon detection

• various concentrations of HAuCl₄ solutions, 5 mM, 10 mM, 20 mM
• 5 mM yielded a pale blue color, 20 mM HAuCl4 solution did not generate

a substantial color change owing to the insufficient quantity of the Na3Ct solution

Result : 10 mM HAuCl4 produced an intense red color within 10 min

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UV-vis spectrophotometry for the detection of LAMP amplicons

• 10 mM HAuCl₄ : the high intensity of the UV absorption peak at 535 nm corresponded to the positive LAMP amplicons owing to the formation of dispersed.
• 5 mM HAuCl₄, : the UV spectrum did not show an intense peak at 535 nm because the AuNPs aggregated with each other.
• 20 mM HAuCl₄ : the positive sample did not yield a peak at 535 nm because AuNPs failed to form.

Multiplex DNA detection with the portable device��

• we designed a simple and affordable poly(methyl methacrylate)-based portable device to detect COVID-19 plasmid DNA and E. faecium spp.
• The positive samples changed from colorless to red in color, and the negative samples remained colorless
• this colorimetric LAMP assay has great potential for end-point detection of DNA in POCT applications because the results are clearly distinguished by the naked eye and do not require any sophisticated instruments.

Conclusion�

• The obtained visual outcomes justified the reliability of the in situ AuNPs that allowed the detection of a minimum quantity of COVID-19 plasmid DNA and E. faecium spp
• The reagents involved in the colorimetric process are readily available and highly stable at room temperature
• Perform the visual detection of infectious diseases in less than 40 min
• The introduced technique was used to detect a variety of DNAs

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