Highly sensitive SERS platform for pathogen analysis by cyclic DNA nanostructure@AuNP tags and cascade primer exchange reaction

Abstract

The capacity to identify small amounts of pathogens in real samples is extremely useful. Herein, we proposed a sensitive platform for detecting pathogens using cyclic DNA nanostructure@AuNP tags (CDNA) and a cascade primer exchange reaction (cPER). This platform employs wheat germ agglutinin-modified Fe₃O₄@Au magnetic nanoparticles (WMRs) to bind the E. coli O157:H7, and then triggers the cPER to generate branched DNA products for CDNA tag hybridization with high stability and amplified SERS signals. It can identify target pathogens as low as 1.91 CFU/mL and discriminate E. coli O157:H7 in complex samples such as water, milk, and serum, demonstrating comparable or greater sensitivity and accuracy than traditional qPCR. Moreover, the developed platform can detect low levels of E. coli O157:H7 in mouse serum, allowing the discrimination of mice with early-stage infection. Thus, this platform holds promise for food analysis and early infection diagnosis.

Keywords: Bacterial detection; Early infection diagnosis; Primer exchange reaction; SERS tags.

Fig. 1

a Schematic illustration of the fabrication of cyclic DNA nanostructure@AuNP tags (CDNA); b Principle of Aptamer-activated cascade PER for signal amplification of E. coli O157:H7; c The principle of target bacteria detection using a CDNA-based SERS platform

Fig. 2

Characterization of the synthesized CDNA SERS tags. a-b Representative TEM images of AuNPs (a) and CDNA (b) (scale bars : 100 nm; scale bar in magnified image: 20 nm); c Zeta potentials of AuNPs, AuNPs-La, AuNPs-Labc and CDNA; d UV–vis spectra of AuNPs, AuNPs-La, AuNPs-Labc and CDNA; e SERS spectra of AuNPs + MB, AuNPs-La + MB, AuNPs-Lab + MB and CDNA; f The Raman intensity and color of CDNA after storage at 4 °C for 1, 5, 10, 15, 20 and 30 days

Fig. 3

Evaluation of biosensor feasibility. a TEM of Fe₃O₄ MNPs and RAuMNPs; b EDS mapping results of RAuMNPs; c The number of E. coli O157:H7 in the supernatant after WMR capture; d PAGE analysis of the feasibility of cPER. L1: DNA marker; L2: AP1; L3: P2; L4: HA; L5: AP1 + HA; L6: P2 + HC; L7: H1 + H2; L8: AP1 + P2 + HA + HB + HC + HD; L9: AP1 + H1 + H2 L10: AP1 + P2 + HA + HB + HC + HD; e Raman intensity of the detection of E. coli O157:H7 with cPER and PER.

Fig. 4

a Raman spectra of E. coli O157:H7 at different concentrations (5 ~ 5 × 10⁵ CFU mL^− 1); b The linear relationship between E. coli O157:H7 concentration and Raman intensity at 1621 cm^− 1; c Linear analysis of E. coli O157:H7 detection by traditional SERS tag (blue) and CDNA SERS tags (orange); d Specificity evaluation of the SERS platform, only E. coli O157:H7 generated obvious Raman signal; e SERS intensity between E. coli O157:H7 and other complicated bacteria samples, the amount of other pathogens was ten times that of E. coli O157:H7; f The Raman intensity of 20 randomly selected SERS spectra acquired from the measurements for 5 × 10⁵ CFU/mL of E. coli O157:H7.

Fig. 5

Measurement of E. coli O157:H7 in real samples. a Raman intensity of pasteurized milk versus contaminated milk; b Raman signal recovery of E. coli O157:H7 spiked in water, milk and human serum; c Ct value for genomic DNA of E. coli O157:H7 in pasteurized milk versus contaminated milk. (paired two-tailed Student’s t test, ****P < 0.0001); d ROC curve analysis of the CDNA-based SERS platform and qPCR.

Fig. 6

Measurement of E. coli O157:H7 in mouse serum. a Schematic representation of the methodology employed to establish an E. coli O157:H7-infected mouse model and subsequent blood collection; b Raman intensity profiles of six mice infected with E. coli O157:H7 and six healthy mice (represented by the broken line) are shown, along with the average trace (indicated by the red and black lines). The error bars represent standard deviations; c Raman intensity of E. coli O157:H7 expressed in serum of infected mice and healthy mice at the indicated times (paired two-tailed Student’s test, *P < 0.05, **P < 0.01, ***P < 0.001); d ROC curve analysis was performed to compare healthy mice (n = 6) with E. coli O157:H7-infected mice (n = 6)