Biosensors and Bioelectronics 132 (2019) 326–332

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Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

New strategy for the gene mutation identification using surface enhanced T
Raman spectroscopy (SERS)
Agata Kowalczyka, Jan Krajczewskia, Artur Kowalikb, Jan L. Weyherc, Igor Dzięcielewskic,
⁎
Małgorzata Chłopekb, Stanisław Góźdźb, Anna M. Nowickaa, , Andrzej Kudelskia
a
Faculty of Chemistry, University of Warsaw, Pasteura 1 Str., PL 02-093 Warsaw, Poland
b
Holy Cross Cancer Center, Stefana Artwińskiego Str. 3, PL 25-734 Kielce, Poland
c
Institute of High Pressure Physics of the Polish Academy of Science, Sokolowska 29/37 Str., PL 01-142 Warsaw, Poland

A R T I C LE I N FO A B S T R A C T

Keywords: An early and accurate diagnosis of a specific DNA mutations has a decisive role for effective treatment.
Conformation analysis Especially, when an immediate decision on treatment most needs to be made, the rapid and precise confirmation
DNA recognition of clinical findings is vital. Herein, we show a new strategy for the gene mutation (BRAF c.1799T > A; p. V600E)
Gene mutation identification identification using highly SERS-active and reproducible SERS substrate (photo-etched GaN covered with a thin
SERS
layer of sputtered gold) and surface enhanced Raman scattering (SERS) spectroscopy. The detection is based on
Surface analysis
the conformation change (gauche → trans) of the alkanethiol linker modifying the capture DNA during the
hybridization process. The value of the intensity ratio of the ν(C–S) bands of the trans and gauche conformer
higher than 1.0 indicated the presence of mutation. The demonstrated new DNA SERS (bio)sensor is char-
acterized by the low detection limit at the level of pg μL–1, wide analytical range from 6.75 pg μL–1 to
67.5 ng μL–1 and high selectivity. The proposed bioactive platforms, based on nanostructured GaN substrates
modified with thiolated ssDNA (single stranded DNA) can be successfully used in the analysis of clinical samples.

1. Introduction biopsy (Khetrapal et al., 2018; Pérez-Barrios et al., 2016). The source of
circulating tumor DNA (ctDNA) are cancer cells that undergo apoptosis
Over the last decade, we have witnessed the tremendous advances (Crowley et al., 2013; Schwarzenbach et al., 2011). ctDNA released
in next generation diagnostics which are a result of developments in from cancer cells is usually fragmented and has a short length of about
modern nanotechnology (Chertow, 2018; Heath, 2015; Jabir et al., 100–200 bp (Diehl et al., 2005; Mouliere et al., 2011). ctDNA represents
2012). The rapid detection of gene mutation, tumor cells or other 1.4–47.9% of circulating free nucleic acids (cfNA) in the blood circu-
cancer biomarkers, which facilitates better disease diagnosis, mon- lation of oncological patients (Leary et al., 2012). ctDNA is present in
itoring and management is especially a major challenge in the field of 50–75% of patients with advanced as well as localized cancer e.g.
personalized medicine, matching patient's needs with appropriate pancreatic, breast, intestine, kidney and brain cancers (Bettegowda
therapeutic strategies that improve the health outcomes due to proper et al., 2014).
treatments. Among oncological patients 90% of deaths are due to me- Circulating free tumor deoxyribonucleic acid (ctDNA) is detected
tastatic cancer (Torre et al., 2015). In the case of solid tumors (lung and tracked primarily based on tumor–related genetic and epigenetic
cancer, colorectal cancer, breast cancer and melanoma) not infre- alterations (Han et al., 2017). The possibility of using ctDNA to detect
quently there are difficulties in obtaining diagnostic material. There- mutations in genes of predictive and prognostic importance for mole-
fore, it is necessary to search for other sources of material that would be cularly targeted therapies has been demonstrated (Oellerich et al.,
easily collected, provide diagnostic data to regularly monitor the ef- 2017). A big advantage of ctDNA analysis is the possibility of earlier –
fectiveness of treatment. A material with such features is peripheral up to 10 months – detection of disease progression in comparison with
blood, which reaches all the cells of the body (Pantel et al., 2009). The radiological methods (Oellerich et al., 2017). The problem in using
tumor releases in the bloodstream tumor cells (CTCs) and nucleic acids ctDNA for diagnostics is the scarce amount of material released into the
(circulating free tumor nucleic acids – ctNA), which are currently being bloodstream and the lack of ultra–sensitive diagnostic methods capable
attempted to adapt to diagnostics in the form of so–called a liquid of detecting 0.1–0.01% of the mutated allele.

⁎
Corresponding author.
E-mail address: anowicka@chem.uw.edu.pl (A.M. Nowicka).

https://doi.org/10.1016/j.bios.2019.03.019
Received 8 February 2019; Received in revised form 8 March 2019; Accepted 11 March 2019
Available online 12 March 2019
0956-5663/ © 2019 Elsevier B.V. All rights reserved.
A. Kowalczyk, et al. Biosensors and Bioelectronics 132 (2019) 326–332

In the early stages of the cancer, only trace levels of biomarkers most cases, the mutation c.1799T > A (p. V600E) is detected (Davies
exist, so they should be detected with high sensitivity and minimally et al., 2002). The gene coding for the BRAF protein is mutated in
invasive medical procedures. Currently, the clinical cancer identifica- 40–70% of papillary thyroid cancers, 50% of melanomas and about
tion is mainly done using imaging techniques, such as X–ray, mam- 10–20% of colon cancers (Domagała and Kowalik, 2014; Hodis et al.,
mography, computed tomography, magnetic resonance imaging, en- 2012; Kowalik et al., 2017; Rutkowski et al., 2014). Therefore, we
doscopy and ultrasonography. Unfortunately, their usefulness in decided to practically test our new approach for identification of a gene
distinguishing between benign and malignant changes is limited mutation (BRAF c.1799T > A; p. V600E) in clinical samples isolated
(Chinen et al., 2015). The existing genomic and proteomic protocols for from tissues and plasma of patients with papillary thyroid carcinoma
ctDNA determination in some body fluids and/or tissues are time and melanoma.
consuming. In consequence, the risk of patients spreading disease
whilst awaiting results increases. Therefore a non–invasive, selective, 2. Experimental setup
ultrasensitive and rapid protocols for early stages of cancer identifica-
tion are still needed. Surface–enhanced Raman scattering (SERS) fulfills 2.1. Materials
those expectations very well. It is one of the ultrasensitive methods,
which can be used for characterization at the molecular level (Chen All chemicals were of the highest purity available. Sodium acetate
et al., 2013; Wu et al., 2015; Zhang et al., 2018). SERS can provide (NaAc; p.a., POCH, Poland), tris(2–carboxyethyl)phosphine hydro-
specific spectroscopic fingerprints of biomolecular structures and chloride (TCEP; Sigma), magnesium acetate (Mg(Ac)2; p.a., POCH,
compositions of tissues, therefore it is a promising technique for the Poland), 2–amino–2–(hydroxymethyl)propane–1,3–diol (Tris; Sigma),
detection and identification of circulating tumor cells. Up to now var- EDTA (Sigma), absolute ethanol (99.8%; POCH, Poland), 1–propa-
ious approaches for SERS sensors for the detection of the specific DNA nethiol (HS–C3H7, Sigma), 4–mercaptobenzoic acid (HS–C6H4COOH,
fragments have been developed (Wang et al., 2010, 2013a) based on: (i) Sigma), 6–mercaptohexan–1–ol (MCH; Sigma). All oligonucleotides
Raman reporter connected to the complementary single stranded DNA – were purchased from MWG–Operon (Eurofins). The following oligo-
after hybridization Raman reporter is moved close to the plasmonic nucleotide sequences were used:
nanostructure and a strong SERS signal is recorded (Cao et al., 2002;
Fabris et al., 2007; Gao et al., 2013; Huang et al., 2015; Peng et al., • probe DNA (5′→3′): thiol–C –CTAGCTACAGAGAAATCTCGAT,
6
2014; Vo-Dinh et al., 2002), (ii) the sensor is composed of a DNA • complementary target DNA (5′→3′): ATCGAGATTTCTCTGTAGC
hairpin chain and a SERS-active plasmonic structure, one end of the TAG,
hairpin probe is covalently bonded to the plasmonic structure, and the • non–complementary DNA (5′→3′): GCTTGACCGGACTGTCCAAGGT
other is tagged with a Raman reporter, in the presence of the specific
DNA target, hybridization between the target and DNA probe disrupts Thiols are strong nucleophiles and unprotected thiols spontaneously
the stem-loop configuration and spatially separates the Raman reporter form disulphides in neutral aqueous solution, therefore the thiolated
from the surface of the plasmonic structure which causes decrease in DNA fragments are synthesized as a disulfide. For reducing the disulfide
the measured SERS signal of the Raman reporter (Wang and Vo-Dinh, bond of thiol–modified oligonucleotides to the active sulfhydryl form
2009; Wang et al., 2013b), (iii) formation of the sandwich-type struc- the probe DNA was dissolved in 200 μL of 10 mM TCEP in TE buffer
tures due to the DNA hybridization reaction and immobilization of (10 mM Tris, 1 mM EDTA, pH 8.0). Then, the solution was mixed in
plasmonic nanoparticles or plasmonic nanoparticles with attached ThermoMixer (Eppendorf) for 60 min at room temperature. Next, the
Raman reporters on various substrates (Chen et al., 2014; Fu et al., 150 μL solution containing 0.3 mol of NaAc and 1 mmole of Mg(Ac)2
2016) including magnetic nanoparticles (Yu et al., 2017) (iv) ag- was added to the mixture. The tube was fill with absolute ethanol,
gregation of the plasmonic nanoparticles induced by target DNA – gently shaked and incubated for 20 min at −20 °C. To isolate the ob-
presence of the specific DNA target cases aggregation of the plasmonic tained precipitate (HS–DNA) from the mixture the solution was cen-
molecules and a significant increase in the intensity of the measured trifuged at 13,000 rpm for 5 min. At the end the pellet was dried at
SERS spectrum, (Wang et al., 2017; Xu et al., 2018), (v) SERS signal of room temperature.
the specific nucleobases normalized to the signal of the phosphate
backbone (Xu et al., 2015), (vi) the detection of point mutations in large 2.2. Clinical samples
DNA fragments utilizing the single-strand conformation polymorphism
technique, utilizing the observation of the large conformational The study material consisted of DNA isolated from FFPE tumor
changes that single- and multiple-base substitutions impose on long tissue and plasma collected from papillary thyroid carcinoma cases and
single-stranded chains (Morla-Folch et al., 2017). cases of malignant melanoma. Isolation was performed using the
In this work we show a new strategy for identification of ctDNA Maxwell® 16 FFPE Plus LEV DNA Purification Kit (Promega) and
using SERS spectroscopy. In our approach the modified at 5′ end with Maxwell® RSC ccfDNA Plasma Kit (Promega) according to the manu-
the hexanethiol linker ssDNA strands, complementary to the target facturer's recommendations. BRAF p. V600E mutation was detected
ssDNA, were self–assembled on the highly SERS–active substrate. The using the Bio–Rad QX100 droplet digital PCR (ddPCR) platform.
recognition layer formed in this way was able to react with the analyte Samples preparation were performed according to manufacturer in-
(target DNA). We found that the conformation of the hexanethiol linker, struction using Droplet PCR Supermix for Probes (No dUTP) (Bio–Rad),
via which the capture ssDNA is attached to the gold surface, depends on wild type primers/probe assay (PrimePCR™ ddPCR™ Mutation Assay:
the presence of the ssDNA fragments complementary to the im- BRAF WT for p. V600E, HEX, Bio–Rad) and mutation primers/probe
mobilized strands in the analyzed sample. In the presence of the com- assay (PrimePCR™ ddPCR™ Mutation Assay: BRAF p. V600E, FAM,
plementary ssDNA fragments the relative surface concentration of the Bio–Rad). The concentration of DNA isolates plasma and tissue ranged
hexanethiol moieties having the trans conformation of the Au–S–C–C from 0.1 to 0.5 and 30–50 ng μL–1, respectively. It should be stressed
chains is significantly higher than when only non–complementary that the number of base pairs in the clinical samples was at least
ssDNA strands are present in the analyzed sample. It means that to 10–fold higher than in the synthetic (target ssDNA).
identify a given ssDNA strand (and hence to identify the DNA muta-
tion), one can use the phenomenon of the hybridization process chan- 2.3. Modification of the SERS substrate with DNA fragments
ging the conformation (gauche–trans) of the thiol chain via which probe
DNA strands are attached to the metal surface. One of the most fre- SERS platforms used in this study were fabricated by nano–s-
quently mutated proteins in the MAPK pathway is the BRAF protein. In tructuring of hetero–epitaxial Si–doped n–type GaN on sapphire

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samples, with free carrier concentration of n = 1·1018 cm–3. The sam- bunch termination of nano–pillars (see Fig. 1C) shows that after sput-
ples were photo–etched in a 0.02 M K2S2O8 + 0.02 M KOH solution tering the plazmonic metal layer is not continuous, but consists of Au
(designated KSO–D in ref. (Weyher et al., 2002)) under a 300 W nano–clusters of less than 100 nm size. The nanostructure nature of the
UV–enhanced Xe lamp (Oriel) illumination in galvanic mode. This GaN substrates allow to use them as a stable, reproducible and highly
procedure was found to be optimal for high enhancement of SERS active SERS platforms. The detailed characterization of this SERS sub-
signal from the test pMBA analyte (Weyher et al., 2019). The surface strate is presented in our previous work (Weyher et al., 2019). The
morphology of the photo–etched samples was examined in scanning average SERS enhancement factor achieved on this substrate was de-
electron microscope (SEM Zeiss Ultra Plus). After etching, thin layer of termined as equal to ca. 2.1·106 (Weyher et al., 2019).
gold was sputtered using a Quorum Q150TS sputter coater with a
function of cleaning oxidized targets. Before SERS measurements the 3.2. DNA–hybridization biosensor with SERS monitoring
samples were cleaned using argon plasma function in Quorum Q150RS
sputterer. In our approach the GaN substrate for surface–enhanced Raman
To modify the SERS substrate by the single– and double stranded scattering experiments, modified with the self–assembled DNA mono-
DNA fragments the following procedure was applied. First, the DNA layer was used for search of the gene mutation. To obtain the in-
solutions (400 ng μL–1 in water) were heated for 10 min at the DNA formation about the amount of thiolated DNA fragments introduced on
melting temperature specified by the manufacturer to protect the single the SERS substrate via Au–S bond the UV–vis spectra of the solutions
stranded DNA fragments from interactions with each other. Next, the containing probe DNA before and after immobilization step were re-
solutions were immediately cooled down in the ice bath and diluted corded, see Fig. 2. From the difference of the obtained absorbance value
with distillated water (Hydrolab, conductivity 0.056 μS cm–1). Next, the at the DNA band (ca. 260 nm), the number of DNA fragments im-
20 μL droplet of probe DNA (69.1 ng μL–1) was placed at the gold sub- mobilized on the SERS substrate was calculated. The surface con-
strate and left under the cover for 2 h at room temperature. The sub- centration of probe DNA on the SERS substrate (5 × 5 mm) was cal-
strate with self–assembled thiol DNA fragments was carefully rinsed culated as 1.38 μg cm–2. To check the ability of the self–assembled
with water to remove non–adsorbed thiol DNA molecules. To prevent thiolated DNA to the hybridization process the absorbance changes of
the irregular adsorption of nucleic bases at the SERS substrate and the solution containing synthetic complementary target DNA was
simply makes the layer uniform (Levicky et al., 1998), the substrate monitored (Fig. 2). On the basis of the decrease in the intensity of the
modified with self–assembled probe DNA was immersed in 1 mM MCH absorption band of target DNA, before and after interaction with probe
aqueous solution. After 1 h the substrate was removed from MCH so- DNA, its surface concentration was determined to be 1.21 μg cm–2. It
lution and gently washed with water. The hybridization process was indicates that almost every immobilized probe DNA fragment is capable
performed at 36 °C by introduction of 20 μL droplet containing to taking part in the hybridization process, the efficiency of this process
67.5 ng μL–1 of complementary target ssDNA on the substrate modified is equal 90%.
with thiolated probe DNA/MCH and left under the cover for 60 min. Detection of the hybridization process was performed using surface
The same procedure was applied in the case of natural samples. The enhanced Raman spectroscopy. Fig. 3 illustrates the representative
scheme of the SERS substrate modification procedure is presented in SERS spectra of GaN substrates modified with ssDNA before (blue line,
Scheme 1A. Fig. 3A-C) and after hybridization process with synthetic target DNA –
fully complementary (red line, Fig. 3A-B), non–complementary DNA
2.4. SERS investigations (green line, Fig. 3C) and DNA isolated from FFPE tumor tissue with
BRAF c.1799T > A; p. V600E mutation (Fig. 3A) collected from papil-
SERS spectra were recorded using a Horiba Jobin–Yvon Labram lary thyroid carcinoma cases and cases of malignant melanoma and
HR800 spectrometer connected to an Olympus BX40 confocal optical plasma without BRAF mutation (Fig. 3B).
microscope equipped with a 50 × long distance objective. Raman The most characteristic Raman bands, which appear in the analyzed
spectrometer was equipped with a diode laser emitting at 785 nm, a 600 wavenumber region (marked in Fig. 3) are bands near 700 cm–1 (at
grooves/mm holographic grating, and a Peltier–cooled CCD detector 640 cm–1 and at 714 cm–1). These bands are due to the ν(C–S) stretching
(1024 × 256 pixel). The SERS substrates modified with a chemisorbed vibration of the alkanethiols adsorbed on the gold surface. The ν(C–S)
capture ssDNA were immersed in the analyzed solution of DNA, and the band at a lower wavenumber (640 cm–1) is characteristic for molecules
SERS spectra were collected for SERS substrates being covered with the having the gauche conformation of the Au–S–C–C chain (see Scheme
analyzed solution. 1B), whereas the ν(C−S) band at a higher wavenumber (714 cm–1) is
characteristic for molecules having the trans conformation of the
3. Results and discussions Au–S–C–C chain, see Scheme 1B (Bryant and Pemberton, 1991a, 1991b;
Kudelski, 2003; Tarabara et al., 1998). Both bands are shifted toward
3.1. GaN substrate characteristics lower wavenumbers in comparison to that of the liquid hexanethiol at
657 and 738 cm–1. This shift can be related to a withdrawal of electron
The quality of the measured SERS spectra is strongly dependent on density from the C–S bond because of bonding of the sulphur atom to
the SERS–activity of the used plasmonic substrate and its reproduci- gold (Bryant and Pemberton, 1991a; Kudelski and Hill, 1999). Although
bility. Therefore, to carry out these DNA SERS studies we used one of the Au–S–C–C fragment of the chemisorbed thiol–modified DNA is only
the most reproducible SERS substrates described in the literature – a very small part of the whole adsorbed system, the vibrations localized
photo–etched GaN covered with the layer of gold (Kaminska et al., at that part of the molecule that directly interacts with the gold sub-
2011). The SERS materials produced by the deposition of films of strate can dominate the measured SERS spectrum because the SERS
plasmonic metals on nanostructured surfaces (as nanostructured sur- enhancement factor decrease significantly with the increasing distance
face of GaN) are excellent substrates for SERS measurements (Lai et al., from the surface of the plasmonic nanostructure (one can predict the
2014). The nano–structures of GaN after photo–etching is shown in r−10 distance dependence of the SERS enhancement factor) (Kennedy
Fig. 1A. The nano–pillars in Fig. 1 were formed on dislocations due to et al., 1999; Stiles et al., 2008).
effective recombination of photo–carriers along the linear defects. This As can be seen in Fig. 3A, in the case of the presence at the GaN
effect was well recognized and was discussed in refs (van Dorp et al., substrate only the probe DNA sequence the intensity ratio of the ν(C–S)
2009; Weyher and Macht, 2004; Weyher et al., 2001). The surface bands of the trans and gauche conformer was equalled about 0.9. After
morphology of photo–etched and of Au–coated samples is shown in hybridization with the complementary synthetic ssDNA strands (target
Fig. 1A and B-C, respectively. The high magnification image of the DNA, red line in Fig. 3A) or with DNA isolated from FFPE tumor tissue

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Scheme 1. A: Scheme of the procedure of modification of SERS substrates by DNA fragments. B: Possible structures of Au–S–C–C chain: trans (T) conformation and
gauche (G) conformation.

Fig. 1. SEM images of GaN samples after KSO–D photo–etching (A) and after sputtering of gold (B–C). Samples tilted 45°.

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Fig. 2. UV–vis spectra of the solutions of 67.5 ng μL–1 probe DNA and com-
plementary target DNA before and after interaction with 10 unmodified and
probe DNA–modified SERS substrates.

with gene mutation (BRAF, c.1799T > A; p.600E; black lines in Fig. 3A)
the relative surface concentration of the hexanethiol moieties having
the trans conformation of the Au–S–C–C chain significantly increases
(the average intensity ratio of the ν(C–S) bands of the trans and gauche
conformer reaches the value above 1). When a non–complementary
DNA fragments were applied, the hybridization process did not take
place. Therefore, no DNA duplex can be formed and no gauche–trans
conformation changes can be detected, see Fig. 3C. The spectra pre-
sented in Fig. 3 are the average from 20 spectra measured at various
areas of the SERS substrate, however, even in a single experiment
carried at a randomly chosen area of the SERS substrate the effect of the
increase in the intensity ratio of the ν(C–S) bands of the trans and gauche
conformer can be clearly observed. Increasing of the relative surface
concentration of the thiolate moieties in trans conformation is typical
for the process of increasing of the arrangement of the formed thiolate
monolayer (Kudelski and Hill, 1999). For some systems we carried out
measurements of 400 SERS spectra at different areas of the sample; the
repeatability was very good; the relative standard deviation was ap-
prox. < 1% and 6.5% before and after hybridization process, respec-
tively. The result of this experiment also shown that the used SERS
substrates are very homogeneous.
The proposed biosensor was tested in the concentration range of the
complementary DNA targets from 0.68 pg μL–1 to 0.37 μg μL–1. With the
increasing concentration of the analyte (target DNA) at the probe
DNA–modified SERS substrate surface the increase of the intensity ratio
of the ν(C–S) bands of the trans and gauche conformer is observed. Fig. 4
illustrates the obtained dependence Itrans/Igauche = f(Ctarget ssDNA),
which was linear in the DNA concentration range from 6.75 pg μL–1 to
67.5 ng μL–1. The detection limit (LOD) was determined from the low
concentration linearity range of the calibration curve according to the
equation:
3σ
LOD =
a (1) Fig. 3. SERS spectra of gold–covered nanostructured GaN substrates modified
with ssDNA (blue lines) after interaction with DNA isolated from FFPE tumor
where σ is the standard deviation of the response of the blank (Itrans/ tissue with detected mutation (BRAF, c.1799T > A; p.600E) (A), DNA isolated
Igauche for GaN substrate modified with thiolated probe ssDNA before from plasma patients without detected mutation (BRAF, c.1799T > A; p.600E)
hybridization process) and a is the slope of the calibration curve. The (B) and non–complementary synthetic DNA (C). Red lines: fully complementary
determined limit of detection (LOD) was ca. 0.17 pg μL–1. target DNA (synthetic); black lines: clinical samples; green line: non–-
complementary DNA (synthetic). (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article).
3.3. BRAF c.1799T > A (p.V600E) mutation SERS identification

We decided to verify whether the process of the rearrangement of SERS clinical sensors for detection of a circulating tumor DNA frag-
the structure of the linkage moiety via which the probe ssDNA is at- ments. To carry out this experiment we used 17 various clinical sam-
tached to the metal surface may be used as an indicator of the hy- ples: 10 samples from patients having BRAF mutation, it means con-
bridization, and hence, may be used as a principle of operation of the taining ssDNA complementary to capture ssDNA (nine samples were

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4. Conclusions

Proposed approach provides fundamental insight into SERS detec-

tion of circulating tumor DNA fragments characteristic for BRAF mu-
tation (c.1799T > A). The detection was based on the conformation
changes (trans and gauche) of the thiol linker via which the capture
ssDNA was anchored to the gold surface, caused by the hybridization
process. These changes are directly associated with the amount of the
double stranded DNA at gold–covered nanostructured GaN substrates.
To our best knowledge the conformational changes of thiol linker in the
DNA identification were used for the first time. The demonstrated new
DNA SERS (bio)sensor is characterized by the low detection limit at the
level of pg·μL–1, wide analytical range from 6.75 pg μL–1 to 67.5 ng μL–1
and high selectivity. The most prominent achievement of this work is
that the proposed bioactive platforms, based on nanostructured GaN
substrates modified with thiolated ssDNA can be successfully used in
Fig. 4. Calibration plot based on SERS data. Inset: Exemplary SERS spectra of the analysis of clinical samples. The constructed DNA SERS sensor has
gold–covered nanostructured GaN substrates modified with ssDNA after hy- been tested on 17 clinical samples (on 10 samples from patients having
bridization with fully complementary synthetic target DNA in various con-
BRAF mutation: nine with melanoma and one with thyroid cancer, and
centration.
on 7 samples from patients without BRAF mutation). We believe that
this simple approach has a great potential in medical diagnostics.
Table 1
Results of genotyping by ddPCR and Itrans/Igauche ratios at gold covered na-
nostructured GaN substrates modified with ssDNA before and after hybridiza- CRediT authorship contribution statement
tion with fully complementary target DNA (dsDNA) and clinical samples gen-
otyped by ddPCR. Agata Kowalczyk: Investigation, Writing - original draft, Writing -
review & editing. Jan Krajczewski: Investigation. Artur Kowalik:
Sample ddPCR Itrans/Igauche
Resources, Writing - original draft, Writing - review & editing. Jan
ssDNA synthetic 0.94 L. Weyher: Conceptualization, Investigation, Writing - original draft,
target ssDNA synthetic 1.54 Funding acquisition. Igor Dzięcielewski: Investigation.
BRAF Thyroid (tissue) Małgorzata Chłopek: Investigation. Stanisław Góźdź:
40/18 mutation p.V600E 1.13
5242/17 no mutation 0.96
Conceptualization. Anna M. Nowicka: Conceptualization,
BRAF Thyroid (plasma) Methodology, Writing - original draft, Writing - review & editing,
450 T/A no mutation 0.93 Supervision. Andrzej Kudelski: Conceptualization, Writing - original
86 T/A no mutation 0.91 draft, Writing - review & editing, Funding acquisition.
BRAF Melanoma (tissue)
1368/16 mutation p.V600E 1.56
2841/16 mutation p.V600E 1.09 Acknowledgments
2292/16 mutation p.V600E 1.18
42/17 mutation p.V600E 1.13
3185/17 mutation p.V600E 1.15 This work was supported by the National Science Centre of Poland
5526/17 no mutation 0.90 Grant no. 2015/19/B/ST8/02004. Andrzej Kudelski thanks the Faculty
4442/17 mutation p.V600E 1.09 of Chemistry, University of Warsaw for its financial support.
4647/17 mutation p.V600E 1.04
4770/17 no mutation 0.85
BRAF Melanoma (plasma) Ethics statement
147/17 mutation p.V600E 1.26
4894/17 mutation p.V600E 1.13
All of the study procedures were approved local Bioethics
909/17 no mutation 0.90
186/18 no mutation 0.96 Commission (No. 16/2014) at Jan Kochanowski University in Kielce
and performed according to the Declaration of Helsinki. All patients
provided signed, informed consent before enrolling in the study.
from patients with melanoma and one sample was from patient with
thyroid cancer) and 7 samples from patients without BRAF mutation. As Notes
can be seen from Table 1, when the clinical sample contained ssDNA
complementary to the capture ssDNA, the intensity ratio of the ν(C–S) The authors declare no competing financial interest.
bands of the trans and gauche conformer was equal to at least 1.04,
whereas for samples that did not contain ssDNA complementary to the
capture ssDNA, this ratio was equal to maximum 0.96. It means that the Declaration of interest statement
observed by us process of the rearrangement of the structure of the
linkage moiety via which the capture ssDNA is attached to the metal Statement 1: The manuscript, or its contents in some other form,
surface may be used as the indicator of the hybridization. Hence, the has not been published previously by any of the authors and/or is not
determination of this process using SERS spectroscopy may be used as a under consideration for publication in another journal at the time of
principle of the operation of the DNA SERS sensors. The SERS results submission.
were in perfect agreement with the results obtained by applying ddPCR Statement 2: All authors have seen and approved the submission of
for clinical samples genotyping. the manuscript.
Statement 3: All of the sources of funding for the work described in
this publication are acknowledged.
On behalf of all co-authors
Anna M. Nowicka

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