Raluca-Ioana Stefan-van Staden*ab,
Alexandru Adrian Bratei
abc,
Ruxandra-Maria Ilie-Mihai
a,
Damaris-Cristina Gheorghe
a,
Bianca Maria Tuchiu
a and
Simona Gurzu
c
aLaboratory of Electrochemistry and PATLAB, National Institute of Research for Electrochemistry and Condensed Matter, 202 Splaiul Independentei Str., 060021, Bucharest-6, Romania. E-mail: ralucavanstaden@gmail.com; Fax: +40213163113; Tel: +40751507779
bFaculty of Chemical Engineering and Biotechnologies, Politehnica University of Bucharest, Bucharest, Romania
cDepartment of Pathology, George Emil Palade University of Medicine, Pharmacy, Sciences and Technology, Targu-Mures, Romania
First published on 11th August 2023
Two miniaturized electrochemical devices were designed for the simultaneous bioanalysis of MMR (MLH1, MSH2, MSH6, PMS2), and of KRAS in whole blood, urine, saliva, and tumoral tissues. The devices comprised besides the electronic part of the potentiostat a combined 3D stochastic microsensor (combined microplatform) with the sensing part based on the modification of graphene decorated with nitrogen, sulfur and boron (NSB-EGR) modified with two types of frutafit: FTEX and FHD. For the assay of MSH2, MSH6, KRAS, and PMS2 higher sensitivities were recorded when the microdevice based on FHD was used, while for the assay of MLH1 the best sensitivity was achieved by using the microdevice based on FTEX. While the limits of quantification for MLH1, MSH2, and PMS2 were not influenced by the modifier, the microdevice based on FHD provided the lowest limit of quantification for KRAS, the microdevice based on FTEX provided the lowest limit of quantification for MSH6. The validation tests performed proved that recoveries of MLH1, MSH2, MSH6, PMS2, and of KRAS in whole blood, urine, saliva, and tumoral tissues higher than 98.50% with RSD (%) values lower than 0.10% were recorded.
MSI status is due to a defective mismatch repair (dMMR) which mostly occurs due to mutations in the MLH1, MSH2, MSH6 and PMS2 genes. Failure in the MMR system function leads to the accumulation of errors within the genome and therefore to tumorigenesis. Another protein related to cancer development is KRAS, which is a GTPase transductor protein responsible for the regulation of cellular growth and differentiation.10 Mutations in the KRAS gene could lead to a continuous activation of KRAS pathway and thus, to cancer development.
Current guidelines recommend dMMR screening for all colorectal cancer patients to identify a potential Lynch syndrome and the patients to benefit from further counseling and genetic testing.11–15 The screening can be done by using immunohistochemistry to evaluate the loss of protein expression16,17 or MSI testing to evaluate unstable microsatellite regions resulting from dMMR.18–20 KRAS also has a very important role in colorectal cancer.21
This paper proposed two miniaturized electrochemical devices for simultaneous assay of MMR (MLH1, MSH2, MSH6, PMS2), and KRAS in whole blood, saliva, urine, and tumoral tissues. The novelty is given by the design of the electrochemical devices used for the fast simultaneous screening tests of biological samples, and by the design of the 3D combined stochastic microsensors, by utilizing a 3D printer to produce the support of the stochastic microsensor, reference sensor, and of the auxiliary sensor; moreover, the composition of the paste (the active side of the stochastic microsensor) is new – the graphene decorated with nitrogen, boron, and sulfur being modified with two types of frutafit: FHD, and FTEX.
The stochastic mode used for all measurements in this paper is based on the channel conductivity.22–24 There is a two-step process: qualitative step – when the MMR and KRAS are recognized based on their signatures (the process taking place is: the molecules enter one by one into the channel, bocking it, and the current drops to zero value – the time spent at this value is the one needed for the molecule to get inside the channel, and therefore it is called the signature of the molecule), and a second step on which the molecule inside the channel is undergoing binding and redox processes (the qualitative step, characterized through the measured ton value – the time needed for the molecule to change its sign during the redox process). The advantages of using the stochastic mode versus other electrochemical methods are the following: the sample does not need any processing before the measurements; the complexity of the matrix does not influence the results of the measurement; the signature is associated with a high reliable qualitative analysis being dependent only on the size, geometry, and velocity of the molecule.
The stochastic microsensor was integrated in a microplatform together with the counter electrode (platinum wire), and the reference electrode (Ag/AgCl electrode) (Scheme 1).
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Scheme 1 Design of the stochastic microsensor, and microplatform of measurement used in simultaneous assay of MLH1, MSH2, MSH6, PMS2, and of KRAS in whole blood, urine, saliva, and tumoral tissues. |
Before and after each measurement, cleaning with deionized water and soft drying with an adsorbant paper were performed. When not in use, the microplatforms were kept in a dry place, at room temperature.
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Fig. 1 Typical diagrams obtained by screening (a) whole blood, (b) saliva, (c) urine, and (d) tumoral tissues with the microplatform based on FHD/NSB-EGR. |
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Fig. 2 Typical diagrams obtained by screening (a) whole blood, (b) saliva, (c) urine, and (d) tumoral tissues with the microplatform based on FTEX/NSB-EGR. |
Combined microplatform based on NSB-EGR and | Signature, toff (s) | Linear concentration range (g mL−1) | Calibration equations; the correlation coefficient, ra | Sensitivity (s−1 μg−1 mL) | LOQ (fg mL−1) | |
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a <C > – concentration = μg mL−1; <ton> = s; LOQ – limit of quantification. | ||||||
MLH1 | ||||||
FHD | 1.2 | 3.20 × 10−16–3.20 × 10−5 | 1/ton = 0.11 + 2.06 × 10−2 × C; r = 0.9995 | 2.06 × 10−2 | 0.32 | |
FTEX | 2.1 | 3.20 × 10−15–3.20 × 10−6 | 1/ton = 0.05 + 1.03 × 10−1 × C; r = 0.9902 | 1.03 × 10−1 | 3.20 | |
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MSH2 | ||||||
FHD | 2.0 | 1.00 × 10−15–1.00 × 10−9 | 1/ton = 0.06 + 2.33 × 102 × C; r = 0.9994 | 2.33 × 102 | 1.00 | |
FTEX | 1.1 | 1.00 × 10−14–1.00 × 10−5 | 1/ton = 0.10 + 37.56 × C; r = 0.9979 | 37.56 | 10.00 | |
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MSH6 | ||||||
FHD | 1.8 | 2.30 × 10−9–2.30 × 10−5 | 1/ton = 0.16 + 1.02 × 10−2 × C; r = 0.9947 | 1.02 × 10−2 | 2.30 × 106 | |
FTEX | 3.4 | 2.30 × 10−15–2.30 × 10−6 | 1/ton = 0.11 + 5.91 × 10−3 × C; r = 0.9907 | 5.91 × 10−3 | 2.30 | |
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PMS2 | ||||||
FHD | 1.4 | 2.70 × 10−15–2.70 × 10−5 | 1/ton = 0.15 + 1.71 × 104 × C; r = 0.9996 | 1.71 × 104 | 2.70 | |
FTEX | 2.5 | 2.70 × 10−15–2.70 × 10−6 | 1/ton = 0.09 + 2.00 × 10−2 × C; r = 0.9949 | 2.00 × 10−2 | 2.70 | |
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KRAS | ||||||
FHD | 1.6 | 2.20 × 10−15–2.20 × 10−5 | 1/ton = 0.06 + 9.50 × 10−3 × C; r = 0.9976 | 9.50 × 10−3 | 2.20 | |
FTEX | 1.3 | 2.20 × 10−15–2.20 × 10−6 | 1/ton = 0.13 + 2.89 × 103 × C; r = 0.9967 | 2.89 × 103 | 2.20 |
Reproducibility and stability studies were performed for each type of combined microplatform. Ten combined microplatforms based on FHD, and on FTEX, respectively, were designed accordingly with the procedure described above. Measurements of the sensitivities were performed for each combined microplatform, and calculations of %, RSD were performed. Values for the %, RSD of the sensitivities calculated were less than 0.27% for the combined microplatform based on FHD while when FTEX was used %, RSD values less than 0.12% were recorded, proving the design’ reproducibility of combined microplatforms. The 20 combined microplatforms' sensitivities were further checked for 30 days in order to establish their stability in time; for all tested combined microplatforms, %, RSD values less than 0.51% were recorded during the 30 days. The variance recorded for measurements performed using both microplatforms when used for simultaneous assay of MLH1, MSH2, MSH6, PMS2, and of KRAS in whole blood, urine, saliva, and tissue samples, did not exceeded 0.10.
A very good correlation between the results obtained using the combined microplatform based on FHD and using the combined microplatform based on FTEX (Fig. 3) were obtained for all samples: MLH1, MSH2, MSH6, PMS2, and KRAS in whole blood, saliva, urine, and tumoral tissue samples.
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Fig. 3 Determination of MLH1, MSH2, MSH6, PMS2, and KRAS in whole blood, saliva, urine, and tumoral tissue samples using the combined microplatforms based on FHD/NSB-EGR, and on FTEX/NSB-EGR. |
The %, RSD values associated to Fig. 3 are shown in Table 2. The values determined shown a high reproducibility of the measurements performed with the combined microplatform.
Combined microplatform based on NSB-EGR and | %, RSD | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
FHD NSB-EGR | FTEX NSB-EGR | ||||||||||
Biomarker | MLH-1 | MSH-2 | MSH-6 | PMS-2 | KRAS | MLH-1 | MSH-2 | MSH-6 | PMS-2 | KRAS | |
Biological fluid | Saliva | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.03 | 0.02 | 0.03 |
Whole blood | 0.02 | 0.03 | 0.02 | 0.02 | 0.02 | 0.02 | 0.03 | 0.02 | 0.02 | 0.02 | |
Tissue | 0.02 | 0.02 | 0.02 | 0.03 | 0.03 | 0.02 | 0.02 | 0.03 | 0.02 | 0.02 | |
Urine | 0.02 | 0.02 | 0.03 | 0.02 | 0.02 | 0.02 | 0.02 | 0.03 | 0.02 | 0.02 |
The paired Student's t-test was performed at 99.00% confidence level for all biomarkers: MLH1, MSH2, MSH6, PMS2, and KRAS. The calculated values for the t-test were lower than 3.21 (tabulated value at 99.00% confidence level is 4.13), proving that there is no significant difference between the results obtained using the two combined microplatforms based on FHD, and on FTEX.
Apart from the t-test, recovery tests of MLH1, MSH2, MSH6, PMS2, and KRAS were performed for whole blood, saliva, urine, and tumoral tissue samples. An initial screening was done to determine the amounts of MLH1, MSH2, MSH6, PMS2, and KRAS in whole blood, saliva, urine, and tumoral tissue samples. Ten different amounts of MLH1, MSH2, MSH6, PMS2, and KRAS were added to the real samples, and the final concentrations were determined. The added amounts of MLH1, MSH2, MSH6, PMS2, and KRAS in whole blood, saliva, urine, and tumoral tissue samples were compared with the found amounts. The results are given in Table 3.
Combine microplatform based on NSB-EGR and | Recovery, % | |||||
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MLH-1 | MSH-2 | MSH-6 | PMS-2 | KRAS | ||
Whole blood | ||||||
FHD | 99.99 ± 0.02 | 99.96 ± 0.01 | 99.83 ± 0.02 | 99.87 ± 0.02 | 99.95 ± 0.02 | |
FTEX | 99.95 ± 0.03 | 99.47 ± 0.01 | 99.91 ± 0.01 | 99.87 ± 0.03 | 99.96 ± 0.02 | |
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Saliva | ||||||
FHD | 99.21 ± 0.03 | 99.21 ± 0.02 | 99.88 ± 0.01 | 99.12 ± 0.03 | 99.77 ± 0.04 | |
FTEX | 99.77 ± 0.05 | 99.30 ± 0.01 | 99.90 ± 0.02 | 95.43 ± 0.04 | 99.43 ± 0.02 | |
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Urine | ||||||
FHD | 99.00 ± 0.02 | 99.20 ± 0.04 | 99.11 ± 0.02 | 99.12 ± 0.02 | 99.18 ± 0.04 | |
FTEX | 99.11 ± 0.04 | 99.22 ± 0.02 | 99.05 ± 0.01 | 99.08 ± 0.03 | 99.21 ± 0.02 | |
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Tumoral tissue | ||||||
FHD | 98.90 ± 0.03 | 98.60 ± 0.03 | 98.77 ± 0.02 | 98.90 ± 0.03 | 98.73 ± 0.01 | |
FTEX | 99.00 ± 0.02 | 98.75 ± 0.04 | 98.97 ± 0.01 | 99.00 ± 0.02 | 98.78 ± 0.02 |
The performed recovery tests show high values for recoveries (all higher than 98.50%) with very low RSD (%), lower than 0.06%, when 10 measurements were performed. Accordingly, high accuracy and precision were achieved when the proposed combined microplatforms were used for the bioanalysis of the samples.
Compared to the results obtained for the assay of KRAS and MLH1, MSH2, MSH6, PMS2,25,26 using stochastic sensors, the working concentration ranges are wider, and the limits of determination are far lower, favorizing the identification and quantification of MLH1, MSH2, MSH6, PMS2, and KRAS in whole blood, saliva, urine, and tumoral tissue samples, at a very early stage of colon cancer.
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