Silicon/Silver nanocomposite-based surface-enhanced Raman spectroscopy (SERS) biosensor for ultrasensitive direct detection of deafness mutations in real system

Surface-enhanced Raman scattering (SERS) is a promising emerging, highly sensitive technique for detecting biological samples. The current research aims to bring an effortless biosensor-based approach to detect and discriminate DNA mutations with their fingerprints for each deafness mutation within the GJB2 (gap junction beta 2) gene. Therefore, we developed silver (Ag) modified silicon-based SERS sensing platform to directly screen GJB2 gene mutations for UAE patients with hereditary hearing loss (HL). The prepared AgNPs@Si biosensor was utilized for establishing a silicon-based SERS biosensing protocol for further analyses toward label-free DNA deafness mutations screening. Furthermore, the comparative studies demonstrated the viability of the developed sensor to distinguish between the normal/mutant samples in the real-time system by investigating the DNA assignments and the peaks intensity. Noticeably, under 785 nm laser excitation distinguishable Raman peaks for cytosine bands (C) are enriched with normal DNA (DMF-34) at 780 cm−1, 1450 cm−1 and 1590 cm−1 whereas with the single nucleotide deletion of cytosine band for the mutant DNA correspondingly predicted with Quenched intensities at the parallel Raman peak positions. The sensitivity studies portrayed the limit of detection to be 1 fg/μL. Further their reproducibility studies evaluation conveyed with low values of % RSD as 10. By the end of this study, we expected to update the mutational profile of the GJB2 gene in the UAE population using the advanced simple SERS-based technique for further supplemental applications in terms of genetic counseling and prenatal diagnosis.


Introduction
Sensorineural deafness is one of the most popularr defect, out of 1000 children there is one child suffering from hearing loss.About 80% of hereditary deafness is contributed by Non syndromic hearing loss (NSHL).Mostly autosomal recessive type mutation is the highest frequency mode of inheritance [1].GJB2 (gap junction protein beta 2) is the most prevalent GJB2 gene among GCC (Gulf Cooperation Council) countries and has been reported that, GJB2 gene accountable for more than 50 % out of all cases with ARNSHL (Autosomal Recessive Non-Syndromic Hearing Loss) in most populations and particularly within UAE (United Arab Emirates) due to high number of the endogamy.This form of high heterogeneity exhibiting Non syndromic hearing loss (NSHL) would increase the challenge to diagnose and screen, despite the results efficiency for the classical methods like Sanger sequencing, DNA microarray, PCR (Polymerase Chain Reaction) and its modifications.These techniques also include high cost and time consumption [2][3][4].From this insight, seeking and developing a rapid, susceptible, and affordable technique to screen such mutations will be of service to improve the diagnosis of deafness mutations in early stages.SERS (Surface Enhanced Raman Spectroscopy) biosensors-based approach put forward a fascinating scope for biological samples as a rapid and sensitive testing method by means of plasmonic nanoparticles based on electromagnetic enhancement (EM).Silver nanoparticles tend to attract and to create an engaging area with great electrical field that can enhance the signal read out of analyte molecules at their proximity.Label free detection of DNA as well mutations are possibly achieved with advanced SERS technology [5,6].
Herein, we aim to fabricate Silicon/silver nanocomposite (SSN) platform for direct screening of hearing loss mutations using Surface enhanced Raman scattering (SERS) platform.The sensor characteristics were detailed as well their sensing mechanisms also projected.The fabricated SSN sensor detected DNA mutations with utmost sensitivity providing the base for SERS to be developed as diagnostic platform in the medical field.

DNA samples preparation
The overall scheme of work (Figure . 1) starts with extraction of DNA from the saliva samples of the affected persons with details as referred to our previous works [7].Two PCR (Polymerase Chain Reaction) tests were conducted to amplify the concentration of DNA samples.It was preceded with (GoTaq® G2 Flexi DNA Polymerase) Promega, REF number M7806, targeting the second exon of GJB2 gene using primers (Cx26 2F and Cx26 2R) followed by Gel electrophoresis confirmation.Next step, purification of DNA was done according to the manufacturer's protocol (Wizard SV Gel and PCR clean-up system, Catalogue number selected: A9281), followed by DNA concentration measurement by Nanodrop One Microvolume UV-Vis Spectrophotometer (Nanodrop One, Thermo Scientific).Finally, all the samples volume were consolidated by dilution with distilled water to end up with 50 μl with a concentration of 10 ng / μl.The extracted, purified DNA samples were stored at -18°C for further use.DNA sample (DMF16) with pathogenic deafness, autosomal recessive hereditary hearing loss which carry mutation with heterozygous state, deletion in the coding region 235delC position 235 (GGGCC) as well (DMF34) normal sequence (GGGCCC) within GJB2 (gap junction protein beta 2) were taken for further analysis with SERS sensing to detect/ discriminate mutations.Moreover, all experimental procedures and informed consents used in this study were approved by the University of Sharjah Research Ethics Committee (No. REC-15-11-P004) and performed in accordance with the relevant guidelines and regulations.

DNA solution preparation for SERS sensing with SSN sensor
SERS sensor (SSN) was fabricated with respect to our earlier reports by facile wet chemical strategy [6].The aqueous DNA stock solutions (DMF 16 and DMF 34) stored in -18°C was taken and thawed.After thawing, SERS studies, 5 μl from each DNA samples were dropped on the top of the SSN sensor substrate for direct detection DNA.All the Raman experiments are done for five times for the assurance of concordance results.

SERS sensor characterization
The SSN sensor was fabricated and characterized for its morphology since it lays on the base for SERS improvement.The sensor exhibited spherical shape with on sight of growth of dumb bells like morphology by the linkage of the adjacent spherical AgNPS by the adopted synthetic strategy (Figure.Detectability studies were evaluated for the fabricated sensor with DMF-16 mutant DNA.The DNA concentration was from 10 ng/µL to 0.001ng/µL were prepared by serial dilutions and subjected to SERS analysis.The DNA features begin are prominent with higher concentration of DMF-16 whereas, the peak intensity and the characteristic peaks were found to be diminished with lower concentrations (Figure .5 a).The sensor detectability from the analysis was found to be 1 fg/ µL.Regression analysis with linear fit provided that it followed the logarithmic linear behaviour with R 2 value being 0.98 (Figure.5 b).

Relative SERS sensing of DNA mutations with normal healthy DNA
In comparison between two DNA sequences, the mutant sample (DMF-16) suffers from heterozygous frameshift mutation caused by base pair deletion in the coding region exon 2 of GJB2 gene (Figure . 6).With SSN sensor sensing, we observed some of the shared peaks within the range of 900 cm -1 -1300 cm -1 and mostly that refer to the DNA backbones vibration, particularly, 920 cm -1 , 1300 cm -1 , and 1600 cm -1 .For the wild type DNA (DMF-34/ GGGCCC) we perceived several distinct peaks, (GGGCCC) within which the prime peaks account for 720 cm -1 , 780 cm -1 , 950 cm -1 , 1080 cm -1 , 1450 cm -1 , 1590 cm - 1 and 1640 cm -1 respectively.The peak assignments are enclosed in Table 1.On the other hand, for the mutant type DNA (DMF-16 / GGGCC) had two featured peaks at Raman shift of 1403 cm -1 and 1605 cm -1 .With reference to the sequence distinctly, the frequency of (C) base pair in the normal sequence (DMF-34) showed as blue color vertical dash lines are highly intense than the mutant sample (DMF-16).The single base pair deletion in DMF -16 was notified with cytosine band quenching at 780 cm -1 , 1450 cm -1 and 1590 cm -1 respectively.The red color vertical dash lines with DMF-16 imparts that it contains only one cytosine band at 1304 cm -1 which normal to appear for any DNA under study with their sequencing.According to the DNA assignment, Raman peaks at 780 cm -1 , 1450 cm -1 and 1590 cm - 1 representing (C) which is predominant with DMF-34 whereas the quenching of the Raman bands at the respective peaks were authenticated with mutant sample DMF-16 also well correlated with PCR sequencing (Figure.In-plane stretching C2=O, C4=O (T)

Conclusion
Congenital hearing loss is one of the prime common sensory disorders over the world and GJB2 gene has been reported to be one of the generality implicated genes within many ethnics group including UAE in terms of recessive non-syndromic hearing loss.Thus, succinctly, in this research we fabricated label-free SERS biosensor (Si/AgNPs-SSN) to detect DNA of healthy person (DMF-34) as well with hearing loss patient DNA mutation (DMF-16) at single site.The SSN sensor detected DNA with utmost sensitivity and the Raman modes of DNA bands were assigned.From the DNA assignment studies clearly, we can state the differences between healthy person and hearing loss patient.However, the study reported is in intermediate level, still it needs some developments to apply for real time diagnosis applications.With the imprinted results we project SERS as a rapid diagnosis tool to screen the hearing loss individuals by the creation of library files from all of the combinations of DNA mutation.

Figure 1 .
Figure 1.Scheme of the DNA extraction and SERS DNA analysis

Figure 2 .
Figure 2. (a) SEM analysis on the morphology of the fabricated SSN sensor (b) Size distribution analysis with Image J software (c) Elemental analysis of the Si/AgNPs nanocomposite SSN sensor.
2 a).The size distribution calculations (Figure. 2 b) with Image J software conveyed that, the average particle size being 75 nm ± 10 nm.Further their elemental analysis confirmation (Figure. 2 c) portrayed the purity of the fabrication process with only the elements of Si and Ag respectively.

Figure 3 :
Figure 3: (a) DMF-16 DNA SERS sensing with SSN sensor (b) DMF -16 DNA sequence obtained after PCR with Sager sequencing (c) Mechanism of SERS DNA sensing The DNA bands of purines and pyrimidines were well explored with utmost sensitivity (Figure.3 a; lower panel).For reference the DNA analyte solution (DMF-16) was also tested with bare Si substrate without plasmonic nanomaterial AgNPs and found to notify only the presence of Si peak at 520 cm -1 (Figure.3 a; upper panel).The DNA sequence (Figure.3 b) obtained from Sanger sequencing after PCR studies matched well with SERS Raman peaks (Figure.3 a).The mechanism of SERS sensing with SSN sensor (Figure.3 c) is contributed by the EM effect amplified with the Plasmonic AgNPs (Figure.4 c).Upon laser exposure, the AgNPs excitation was achieved to create collective oscillation of electron clouds activation its unique property of localized surface plasmon resonance (LSPR).The spherical as well dumb bell conjugation of morphology with less interparticle distance enables high density of electron

Figure 4 :
Figure 4: (a) Mapping spectrum of DMF-16 for peak at 1605 cm -1 (b) RSD analysis of DMF 16 for peak at 1605 cm -1 (c) EM field simulations of fabricated SSN sensor with FDTD.