LEI 11788 PDF

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The analysis of DNA has led to revolutionary advancements in the fields of medical diagnostics, genomics, prenatal screening, and forensic science, with the global DNA testing market expected to reach revenues of USD However, the current methods for DNA analysis remain dependent on the necessity for fluorophores or conjugated proteins, leading to high costs associated with consumable materials and manual labor.

Here, we demonstrate a potential label-free DNA composition detection method using surface-enhanced Raman spectroscopy SERS in which we identify the composition of cytosine and adenine within single strands of DNA. This approach depends on the fact that there is one phosphate backbone per nucleotide, which we use as a reference to compensate for systematic measurement variations.

We utilize plasmonic nanomaterials with random Raman sampling to perform label-free detection of the nucleotide composition within DNA strands, generating a calibration curve from standard samples of DNA and demonstrating the capability of resolving the nucleotide composition.

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The work represents an innovative way for detection of the DNA composition within DNA strands without the necessity of attached labels, offering a highly sensitive and reproducible method that factors in random sampling to minimize error. The evaluation of DNA without costly DNA sequencing 12 or labeling methods 3 is imperative to reducing healthcare costs, so efforts must be made to develop DNA screening technologies that rely on cost reduction technology and scientific simplicity.

Here, we examine a new label-free DNA detection method using surface-enhanced Raman spectroscopy SERS 4 — 6an optical technique that probes molecules directly rather than relying on the use of labels. SERS has been proposed as a label-free detection method of DNA 7 — 9 due to the rich molecular information that Raman scattering reveals 10 — 14 li, but previous attempts have suffered from the inability to quantify information related to the composition of DNA 12oei We report an approach to accurately identify the composition of the DNA bases adenine and cytosine benefiting from the fact that each base shares the same phosphate backbone structure, which can be treated as a reference to extract the base composition of adenine and cytosine within the DNA strands.

This self-reference approach would be immune to varying experimental conditions due to the normalization of the data, as compared to other experiments that rely on absolute intensity measurements in which the signal varies as conditions change. The self-reference approach provides a calculated ratio of the nucleotide signal with respect to the phosphate backbone, regardless of the variation in experimental conditions.

Additionally, a highly accurate and precise statistical distribution can be maintained when a large number of experiments are conducted with uncontrollable environmental conditions via random sampling, which combined with the self-reference approach can be employed to determine the nucleotide composition in the DNA strand. As an example, we employ SERS as a demonstration of the self-reference approach, in which the nucleotide composition lri acquired from several samples of synthesized DNA strands composed of varying mixtures of adenine and cytosine.

In this case, a high concentration of adenine and cytosine is used in order to maximize the SERS signal at short acquisition times. Additionally, as this is the first demonstration of this technique, we selected adenine and cytosine due to the fact that their prominent Raman modes do not overlap with each other or the phosphate backbone mode, and thus are the ideal bases to utilize as a proof of concept.

Future studies will take into account the additional bases of guanine and thymine; however, as a preliminary demonstration, only adenine and cytosine were analyzed in this work. To demonstrate the simplicity of a self-reference approach on readily available substrates, we employ low cost random silver films as plasmonic resonators that localize the electromagnetic field for improved optical signal generation 16 — The self-reference of the phosphate backbone provides statistical bias to eliminate the nature of the experimental variation, which in our case, is caused by the DNA orientation on the metal surface, random distribution of plasmonic particles, and variable signal to noise ratio.

The random silver films used in this work are fabricated in an uncontrolled manner, and thus have an uneven distribution of plasmonic hot-spots. However, as demonstrated in this work, the variation in the electromagnetic field enhancement is significantly reduced by taking into account hundreds of Raman spectra that minimize the input noise into our system.

Because measured SERS spectra vary across similar samples of interest and thus offer poor quantitative information, based on the self-reference strategy discussed above, we introduce a normalization procedure in which the ratio of the ring-breathing-modes of the nucleotides to the backbone mode of the DNA is found for each spectrum. Thus, the normalization procedure is dependent on the backbone mode as it stays constant throughout the spectral measurements.

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As shown in Fig. The current work demonstrates the feasibility of a self-reference approach for the identification of nucleotide composition within DNA using a simplified model, in which synthesized single strands of DNA composed of adenine and cytosine are functionalized to random silver films and the SERS spectra are acquired.

Normalization procedure for DNA composition detection using Raman spectroscopy. There are significant variations in the detected Raman signals across the same sample, which can be attributed to disproportionate distributions of hot spots, inconsistent functionalization of DNA strands, and fluctuations in optical setup. Here, we aim to use random sampling in surface-enhanced Raman spectroscopy to collect sufficient Raman data that provides an accurate representation of the sample population to make appropriate inferences regarding the composition within DNA strands.

Thus, we use random silver films as the SERS substrates, as the substrates produce nanoscale islands that generate localized electromagnetic field enhancement while also providing a random distribution of hot-spots that limits systematic bias.

When qualitatively and quantitatively analyzing the data, it is apparent that there are fluctuations between each individual Raman spectrum due to the random hotspot distribution and variations in the ssDNA functionalization. Thus, relying on a limited number of Raman spectra to extract quantitative information from the system is insufficient for the calculation of nucleic acid composition within DNA strands.

Instead, we use the entire Raman measurement set to accurately represent the system under study. To obtain an accurate representation of the population, we calculate the normalization ratio for each Raman spectrum in the set ofand then plot the probability density function PDF of the normalization ratio based on the measurements.

To confirm the appropriateness of the lognormal distribution fit, we plot the cumulative density function Fig.

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Random Raman mapping of ssDNA funtionalized to silver films. After establishing the necessary constraints to account for variations and fluctuations across samples, we propose to measure the composition within a random strand of DNA by first establishing a calibration curve using several known standards of DNA mixtures. To measure the composition of nucleic acids, ssDNA of bases in length were purchased from Integrated DNA Technologies with the following compositions: To generate the calibration curve for the estimation of composition within DNA using the random Raman sampling procedure, we perform measurements on lwi standard and plot the probability density functions for each standard.

A single Raman spectrum for each standard is shown in Fig. Incorporating measurements and calculating the lognormal distribution provides more accurate information related to the composition of nucleotides in DNA strands in comparison to relying on single measurements, and thus more precise calibration curves can be generated from the Raman measurements.

Raman spectra and corresponding probability distributions for the 5 standards. To reduce the error in the calibration curve, two Raman mapping procedures are performed for each lwi with a total of 10 points median of each lognormal distribution used for 11788 calibration curve. The linear dependence of the cytosine normalization ratio is due to the lack of charge-transfer effect at nm 21 ; lie it is important to note that at shorter wavelengths e.

The calibration curves for both adenine and cytosine can be found in Fig. For adenine, the positive peak intensity is attributed to noise, while for cytosine, the positive peak intensity is caused by both noise and the existence of the phosphate skeleton stretching Raman mode of DNA which overlaps with the cytosine ring-breathing-mode.

A discussion and example spectrum demonstrating the 1178 of the non-zero y-intercepts can be found in S2. Thus, the LOD for adenine and cytosine are compositions of 9. To determine the composition of a random strand of 117888, we used a randomization algorithm to generate a random sequence of DNA containing bases of both adenine and cytosine.

Probability density functions and calibration curve for detection of composition within random ssDNA. This is lower than the average peak intensities of the A 111788 C ring-breathing-modes for the SERS spectra set, which are a. Thus, the surface-enhanced affect improves the signal to noise ratio and also reduces the variation 11878 the measured signals, allowing for a lower standard error in the calibration curves. To visualize this difference, Fig. The larger variances of NRS 1.

This also suggests that incorporating a SERS substrate with a greater enhancement will reduce the variance and minimize the standard error. Thus, we have demonstrated that our method is a eli improvement over performing these measurements using normal Raman scattering without enhancement from a SERS substrate.

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To further improve our method to achieve single nucleotide resolution in the future, we propose to implement high local field enhancement factor nanoplasmonic resonators 22 — 24 that will greatly enhance the signal to noise ratio of the system. Despite the cost efficiency of random silver films, they are poor SERS substrates due to their weak local field enhancement with LFE estimations between 0.

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We can aim for the utilization of resonators capable of highly confining the local electric field, which will increase the LFE from a maximum of 2. Using plasmonic resonators capable of LFE between 10 to would drastically increase the signal to noise ratio, which in turn would reduce our standard error to a level capable of achieving single nucleotide resolution.

In this work, we demonstrated label-free detection of the adenine and cytosine composition within DNA strands using a random Raman mapping procedure. Single strands of DNA composed of adenine and cytosine were functionalized to random silver films, and Raman measurements were acquired for 5 different standards. The normalization ratio of the adenine and cytosine to backbone modes were calculated for each set of measurements, and the probability density functions were calculated which fit lognormal distributions.

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Calibration curves were generated using the standards, and the nucleotide composition of a random sequence of ssDNA was estimated. Thus, this work shows promise in implementing this novel method as a technique in determining the composition of bases within DNA strands without the use of labels.

While the work discussed here is promising, additional method development is necessary to realize eventual application for DNA composition detection. This work focused on adenine and cytosine as a proof of concept, so future direction will consist of determining the composition of all bases within random strands of DNA. Additional future developments will incorporate higher local field enhancement nanoplasmonic resonators that will increase the signal to noise ratio, reduce variation in the Raman spectra, and minimize standard error in the calibration curve.

Eventually, the aim of the technique is to utilize this method for future label-free studies of DNA, in which distinctions can be made on changes to the DNA composition without the need to modify the DNA using standard techniques such as labeling assays. A table of the sequences for each ssDNA standard can be found in the supplementary information.

The DNA concentration selected was chosen to ensure sufficient coverage, and the corresponding salt ratio was used to neutralize the phosphate backbone and promote attachment of the nucleotides directly to the silver.

After acquiring the measurements, cosmic ray removal and baseline subtraction using the Intelligent Fitting function were performed using the Renishaw WiRE 4.

Additional information on the fitting models can be found in the supplementary information. A description of the methods, table of the ssDNA sequences, lognormal distribution plots 11788 additional measurements, and statistical calculations are available in supporting information.

This material is available free of charge via the Internet at http: All authors discussed the results and commented on the manuscript. Supplementary information accompanies this paper at Springer Nature remains neutral with regard to 1188 claims in published maps and institutional affiliations. National Center for Biotechnology InformationU. Published online May 9. FreemanLin Pangand Yeshaiahu Fainman. Author information Article notes Copyright and License information Disclaimer.

Received Oct 25; Accepted Apr To view a copy of this license, visit http: Abstract The analysis of DNA has led to revolutionary advancements in the fields of medical diagnostics, genomics, prenatal screening, and forensic science, with the global DNA testing market expected to reach revenues of USD Introduction The evaluation of DNA without costly DNA sequencing 12 or labeling methods 3 is imperative to reducing healthcare costs, so efforts must be made to develop DNA screening technologies 1178 rely on cost reduction technology and scientific simplicity.

Open in a 117888 window. Results There are significant variations in the detected Raman signals across the same sample, which can be attributed to disproportionate distributions of 11788 spots, inconsistent functionalization of DNA strands, and fluctuations in optical setup. Table 1 Median eli the lognormal distributions for each standard and the random sample. Electronic supplementary material Supplementary Information 3.

Notes Competing Interests The authors declare no competing interests. Footnotes Electronic supplementary material Supplementary information accompanies this paper at Shendure J, Ji H.

Branton D, et al. The potential and challenges of nanopore sequencing. Fluorescent in situ sequencing on polymerase colonies. Anomalously intense Raman spectra of pyridine at a silver electrode. Theory of Raman scattering by molecules adsorbed on electrode surfaces. Ed 50— Barhoumi A, Halas NJ.