Human ACE2-Functionalized Gold “Virus-Trap” Nanostructures for Accurate Capture of SARS-CoV-2 and Single-Virus SERS Detection

Highlights Extremely sensitive and prompt COVID-19 SERS biosensor to detect SARS-CoV-2 virus in the contaminated water at single-virus level. SERS sensor features a low detection limit of 80 copies mL−1 for the simulated contaminated water by SARS-CoV-2 virus as short as 5 min. ACE2-modified SERS sensor with machine-learning and identification standard enable rapid detection of novel yet-unknown coronaviruses. Supplementary Information The online version contains supplementary material available at 10.1007/s40820-021-00620-8.


S1 SERS Enhancement Factors of GNAs
The samples for SERS measurement were prepared by soaking the sample into R6G ethanol solutions with different concentrations for 2 h, then rinsing in ethanol and drying in a gentle N2 flow. And Raman spectra of 1 mM and 0.1 M R6G adsorbed on silicon substrates, 0.1 mM R6G adsorbed on Au film, 1 nM and 0.1 nM R6G adsorbed on GNAs were shown in Fig. S1. To estimate the enhancement factor for our nanoneedles arrays, the total SERS substrate EF (Enhancement Factor) was calculated for R6G on the Au nanoneedles samples according to the equation as follows: where Isurf and Ibulk denote the integrated intensities for 1365 cm -1 band of the 0.1 nM R6G adsorbed on the GNAs and 0.1 M R6G on silicon respectively. Whereas Nsurf represent the corresponding total number of R6G molecules excited by the laser beam, which include molecules adsorbed on Au nanoneedles and adsorbed elsewhere on the surface in between Au nanoneedles. Nbulk represent the average number of R6G molecules on silicon in the scattering volume for the Raman measurement. As for the numerator ratio of molecules in a in a Ramanfocused window on the GNAs and silicon substrates is shown in follows: = = 3.14 × 1 2 + 9 × 3.14 × 0.15 2 + 3.14 × 0.3 × 0.5 × 0.7 × 9 × 3.14 3.14 × 1 2 = 3.8 In this formula, the diameter of the nanoneedles in the stem part is around 300 nm, and the density of the nanoneedles is approximately 9 μm -2 . The axis lengths are around 700 nm, and the lengths of the nanoneedles is 300 nm. The diameter of the focused area for Raman measurements is 2 . Therefore, the number of molecules in a in a Raman-focused window on the GNAs substrates is 3.8 times that of silicon substrates for the same concentration of probe molecule solution, the value of ⁄ is determined to 3.8 × 10 −8 . Therefore, it is noted that an enhancement factor of above 3.9×10 9 for GNAs was obtained relative to silicon substrates.  . The higher affinity of SARS-Cov-2 S (10-to 20-fold than that of SARS-CoV) for Human ACE2 may contribute to the significant ease with which SARS-CoV-2 can spread from person-to-person [S1].
Surface protein and lipid profiles are distinctive characteristics of each virus. Usually, viruses can generate detectable characteristic Raman signals containing the surface protein and lipid profiles when their individual surface molecules are adequately in contact with metal nanoparticles, because viruses possess unique surface protein and lipid profiles on their outer layer [4,5]. SARS-CoV-2 viruses are approximately 100 nm in diameter and thereby larger than the molecules that are conventionally analysable by SERS. In the case of SARS-CoV-2, the spike glycoprotein (S protein) on the virion surface mediates receptor recognition and membrane fusion [S1, S3]. CoV-2 virus particles are covered by S proteins with the size of several nanometer. When their individual surface of SARS-CoV-2 virus are adequately in contact with metal nanoparticles, the surface S proteins will range in the "hot-spots" area and generate detectable characteristic Raman signals. Therefore, the detectable characteristic Raman signals usually contained the surface S protein will tend to dominate the SERS-Raman spectra of coronavirus. Consequently, we believed that real-time coronavirus detection can be achieved by measuring the Raman signals from the surface molecules of coronavirus. It is worth noting that the application of dropping method is because GNAs without ACE2 modification have little affinity with SARS-CoV-2 S protein, and can't obtain the Raman signals for such-low concentration R6G by immersing method.
When the SARS-CoV-2 S protein solution was further diluted to 6.33× 10 −10 M, The Raman detecting of SARS-CoV-2 S was conducted by dropping the diluted protein solution of 6.33× 10 −10 M (LOD) on ACE2-modified GNAs (5 mm×5 mm), the number of virus ( 2 ) in a Raman-focused window without enrichment of virus is shown below: 2 = 10 × 10 −6 × 6.33 ×10 -10 × 6.02 ×10 23 × 3.14 × 1 2 5×5 2 ≈500 Therefore, the enrichment multiple is ≥ 500 1.7×10 −4 =2.94× 10 6 , assuming the same number of molecules for LOD can be detected by immersing ACE2-modified GNAs and dropping on GNAs without ACE2-modification. It indicates that the S proteins can be enriched (10 6 -fold) on GNAs by ACE2-modification.    S8. a SERS spectra of healthy girl's urine and VS in urine (2200 copies/mL). b 2D PCA plots for the Raman spectra from pure urine and VS in urine. Red dots and blue squares represent the projected Raman spectra for the pure urine and VS-positive urine. c The Raman intensity mapping from pure urine (right) and VS-positive urine (left) at different frequency positions. d Loading of the PC1 and PC2 for the spectra from pure urine and VS-positive urine. The green line represents the loading of PC1, and the purple line represents the loading of PC2. The PCA result shows that the SERS for urines are uniquely projected to the left for negative urine (PC1 < 0, Fig. S7-b) and right for VS-positive urine (PC1 > 0, Fig. S7-b). The pink and blue bands indicate the characteristic Raman shifts for pure urine and VS-positive urine, respectively.

Fig. S10
DA results to identify 6 Vs-containing adult urines, 6 Vs-containing chronic nephritis' urines with the viral load of 2200 copies/mL, and 9 Vs-containing adult urines with the viral load of 220 copies/mL

VS-containing Urine with the Viral Load of 220 Copies/mL
The SERS chips (0.5×0.5 mm 2 ) was conducted to collect the Vs virus by operation as Fig. S8. VS-containing urine of 200 mL (stimulated the volume of normal adult urine each time, about 100-300 mL) with the viral load of 220 copies/mL were operated 2-3 cycles to enable the virus bind with SERS-chips as completely as possible. The diameter of the focused area for Raman measurements is 2 . Therefore, the maximum of average distributed virus particles in a Raman-focused window is as follow: