Electronic Supplementary Information · 2019. 11. 18. · Electronic Supplementary Information...

Electronic Supplementary Information

Polymerization-driven successive collapse of DNA dominoes enabling

highly sensitive cancer gene diagnosis

Haiyan Dong,a* Bo Lu,a Jie Wang, c Jingjing Xie,d Kuancan Liu, e Lee Jia, c Junyang Zhuangb*

a. Fujian Key Laboratory for Translational Research in Cancer and Neurodegenerative Diseases, Institute for Translational Medicine, Fujian Medical University, Fuzhou, Fujian 350108, China.*E-mail: [email protected] (H. Dong)b. The Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian Province, School of Pharmacy, Fujian Medical University, Fuzhou 350122, China.*E-mail: [email protected] (J. Zhuang)c.Institute of Oceanography, Minjiang College, Fuzhou, Fujian 350116, China. d. School of Pharmaceutical Sciences, and Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiang'an South Road, Xiamen, Fujian 361102, China. e.Institute for Laboratory Medicine, 900 Hospital of the Joint Logistics Team or Dongfang Hospital, Fuzhou, Fujian, 350025, China.

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Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2019

EXPERIMENTAL SECTION

Materials and chemicals

The phi29 DNA polymerase and KF exo- polymerase (KFE) including 10×Reaction

buffer were ordered from New England Biolabs (USA) Ltd. SYBR Green I dye that is

10,000-fold concentrated solution dissolved in dimethyl sulphoxide was purchased

from Dingguo Biochemical Reagents Company (Beijing, China), while the working

solution was prepared by diluting the stock solution with ultrapure water. The Large

fragment Escherichia coli DNA polymerase I (KFL) including the 10×Reaction

buffer, DNA ladder (DL 500), The mixture of deoxyribonucleoside 5’-triphosphate

(dNTPs) and Animal Cell Genomic DNA exaction Kit were purchased from Takara

biotechnology Co. Ltd. Tumor tissue and human blood samples were collected and

donated by the 900 Hospital of the Joint Logistics Team or Dongfang Hospital

(Fuzhou, China). All other chemical reagents were of analytical grade and used as

received unless otherwise stated. Ultrapure water, purified by a Kerton lab MINI

water purification system (UK) (resistance > 18 MΩ/cm), was used to prepare all

aqueous solutions throughout the experiments in this study.

All oligonucleotides used in this work were synthesized with HPLC purification by

Sangon Biotech (Shanghai) Co., Ltd (China). The sequences of these oligonucleotides

were listed in Table S1, and their secondary structures were checked by the “mfold”

program (Http://mfold.rna.albany.edu/). All oligonucleotide stock solutions were

prepared in 1×TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6) and stored at 4

before use.Table S1. Sequence of oligonucleotides designed in this study.

Name Sequence (5' to 3') description a

Quencher Probe (Q-LP)

Fluorescence Probe (F-HP)

5'-CACTCACTGTCACAt(DABCYL)CACGTCGTCGAGAAGCGGA CTGCACACTTAATTAT-3'5'-GACGTGAt(FAM)GTGACAGTGAGTGGTGTGCAGTCCGCTTCTCGAAcgagctcCACAAACACGCACCTCAAAGgagctcgCGT GC-3'

P1 (MB primer1)P2 (MB primer2)

5'- TTTT GCA CGC GAG CT-3'5'- TTTTTT GCA CGC GAG-3'

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http://mfold.rna.albany.edu/)

P3 (MB primer3)P3a (MB primer4)P3b (MB primer5)P3c (MB primer6)P4 (MB primer7)

5'- TTTTTTTT GCA CGC G-3'5'- TTTTTTTTT CA CGC G-3'5'- TTTTTTTTT A CGC GA-3'5'- TTTTTTTTT GCA CGC-3'5'- TTTTTTTTTT GCA CG-3'

Target DNA (p53 gene)MT1 (Mutant target DNA1)MT2(Mutant target DNA2)MT3 (Mutant target DNA3)MT4(Mutant target DNA4)MT5(Mutant target DNA5)MT6(Mutant target DNA6)MT7(Mutant target DNA7)

5'-CAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTG-3'5'-CAGCTTTGAGGTGCaTGTTTGTGCCTGTCCTG-3'5'-CAGCTTTaAGGTGCGTGTTTGTGCCTGTCCTG-3'5'-CAGCTTTGAGGTGCGTGTTTaTGCCTGTCCTG-3'5'-CAGCTTTGAGGTGCaTGTTTtTGCCTGTCCTG-3'5'-CAGCTTTGAGtTGCaTGTTTtTGCCTGTCCTG-3'5'-CAGCTTTtAGtTGCaTGTTTtTGCCTGTCCTG-3'5'-CAGtTTTtAGtTGCaTGTTTtTGCCTGTCCTG-3'

Target DNA 1 (T1)Mutant DNA1 (M1)

5'-TTTGAGGTGCGTGTTTGTGCCTG-3'5'-TTTGAGGTGCaTGTTTGTGCCTG-3'


5'-GAGGTGCGTGTTTGTGCCTG-3'5'-GAGGTGCaTGTTTGTGCCTG-3'


5'-GGTGCGTGTTTGTGCCTG-3'5'-GGTGCaTGTTTGTGCCTG-3'


5'-GTGCGTGTTTGTGCCTG-3'5'-GTGCaTGTTTGTGCCTG-3'


5'-GCGTGTTTGTGCCTGTCCTG-3'5'-GCaTGTTTGTGCCTGTCCTG-3'


5'-GTGTTTGTGCCTGTCCTG-3'5'-aTGTTTGTGCCTGTCCTG-3'

The underlined and bold italic fragments in F-HP can hybridize with the same

formatted region in Q-LP. The lowercase sequences in F-HP are the stem region of

hairpin structure. The italic sequence in the primers can bind to the italic sequence at

the 3' terminus of F-HP. The gray sequences indicate the matching bases between F-

HP and different target DNA (including the mutant ones), and the lowercase letters

among the gray sequence indicate the mutant bases.

Apparatus

Fluorescence measurements were performed on a Hitachi F-7000 fluorescence

spectrometer with a Xenon lamp as the excitation source (Hitachi, Ltd., Japan)

controlled by FL Solution software. The excitation wavelength of 492 nm was used

and the emission spectrum between 500 to 600 nm was collected. The fluorescence

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value at 518 nm was recorded for the quantification of target gene. Both of the

excitation and emission slits were set at 5.0 nm with a PMT Voltage at 600 V.

The agarose gel electrophoresis (or native-PAGE gel electrophoresis) experiment

was conducted with a constant voltage at 80 V at a gel electrophoresis instrument

(BIO-RAD, USA). The images were collected by a ChemiDoc XRS + imaging

system with Image Lab image acquisition and analysis software (BIO-RAD, USA).

The DNA ladders were stained by the Gen Green dye.

Target p53 gene detection by PDDC system

The p53 gene was used as a model analyte in this work. We employed the

developed PDDC system to detect p53 gene and their mutations in the following steps.

Firstly, 35.5 µL 1×Reaction buffer, 3.5 µL of 10 µM Q-LP, 1.5 µL of 10 µM F-HP

were thoroughly mixed and heated at 90 for 5 min, followed by cooling down to

room temperature. The mixture was then stored at room temperature for 1.5 h to

enable the formation of DNA dominoes. Subsequently, 5 µL of p53 gene with

different concentrations and 2 µL of 10 µM primer (P3a), 1 µL of 10 mM dNTPs, 0.5

µL of 5 U/µL KFE polymerase were injected to the DNA dominoes and incubated at

37 for 2 h. The volume of final reaction solution is 50 µL. Finally, the

polymerization reaction was terminated by heating to 80 for 20 min as

recommended by the indicator. After adding 150 µL of 1×Reaction buffer, the

fluorescence spectrum was collected. The difference of the fluorescent intensity

between the target sample and blank was used to characterize the assay performance.

To evaluate the specificity of PDDC system, the mutant p53 DNA with one or more

base mutations were used instead of wild-type p53 DNA and assayed using the

aforementioned method. The specificity of PDDC system was estimated based on the

intensity difference between the fluorescence signal triggered by the mutant p53 DNA

and the signal triggered by the wide-type target p53. The concentrations of target p53

gene samples were calculated from 50 µL reaction solution.

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Statistical analysis

All the experiments were repeated three times. The error bars are standard

deviations of measurements. The statistical analysis represents the means ± SD of

three independent measurements as compared with the control (*P < 0.05, **P <

0.01).

Live subject statement

All clinical samples in this experiment were obtained from the 900 Hospital of the

Joint Logistics Team with the authorization of patients and all experiments were

complied with the ethical regulations of the 900 Hospital of the Joint Logistics Team

(Fuzhou, China) and Fujian Medical University. The authors also state that informed

consent was obtained for any experimentation with human subjects. This protocol was

approved by the Medical Ethics Committee of 900 Hospital of the Joint Logistics

Team and the Medical Ethics Committee of Fujian Medical University.

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Optimization of experimental conditions

In order to achieve the optimal analytical performance, several experimental

conditions including the sequence of polymerization primer, the species of

polymerase used and the enzymatic reaction time, were investigated. The F/F0 value

(where F and F0 respectively represented the fluorescence intensity in the presence

and absence of target DNA) was adopted to evaluate the assay performance of the

developed method. To explore the optimal polymerization primers, 7 different

polymerization primers were designed and tested (as listed in Table S1). As shown in

Fig. S1, a maximal F/F0 value was achieved when the primer named as p3a was used

in assay system. Therefore, we chose p3a as the primer for the polymerization

reaction. Next, to select an optimal polymerase for the polymerization reaction in the

developed assay system, we tested the signal generation performance of 3 different

polymerases including phi29 DNA polymerase, KF exo- polymerase (KFE) and large

fragment Escherichia coli DNA polymerase I (KFL). As shown in Fig. S2, the

maximum F/F0 value was achieved when the KFE was employed to catalyze the

polymerization reaction. Hence, KFE was used in the developed assay system. As

shown in Fig. S3, the F/F0 value was found to increase with the increasing enzymatic

reaction time, and reached a maximal ratio value at 2 h. The longer reaction time did

not obviously change the F/F0 value. More notably, when the reaction time increased

to more than 2 h, the background signal increased substantially. The main reason

might be that the stem-loop structure of a small number of F-HP loosed after

incubating with the primer for a long time, which caused nonspecific polymerization

reaction and fluorescence signal. Thus, we chose 2 h as the enzymatic reaction time.

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Fig. S1. (A) The fluorescence intensity (F) at 518 nm corresponding to target DNA and

background fluorescence signal (F0) of the sensing system; (B) The relationship between the

signal-to-background ratio (F/F0) and the primers. The concentration of target DNA was 100 nM.

Fig. S2. (A) The fluorescence intensity (F) at 518 nm corresponding to target DNA and

background fluorescence signal (F0) of the sensing system using different kinds of polymerases;

(B) The signal-to-background ratios (F/F0) of the sensing system using different kinds of

polymerase. The concentration of target DNA was 100 nM.

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Fig. S3. (A) The effect of polymerization reaction time on the fluorescence intensity (F) at 518 nm

responding to target DNA and the background fluorescence signal (F0); (B) The relationship

between the signal-to-background ratio (F/F0) and the polymerization time. The concentration of

target DNA was 100 nM.

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Extraction and characterization of genomic DNA from cell lines, tumor tissue

and whole blood

The genomic DNA of cancer cell lines, oesophageal carcinoma tissue and whole

blood was extracted using the TIANamp® Genomic DNA Kit (TIANGEN BIOTACH

CO., LTD, Beijing) according to the supplier's instructions. Listed below are the

detailed operation procedures (taking cell lines for example):

(1) The human colorectal cell lines (LOVO for wild-type p53 and SW620 for

mutant p53) were cultured at 37 in Ham’s F12K medium supplemented

with 10% fetal calf serum, 100 units/mL penicillin and 100 µg/mL

streptomycin. When cultured to desired confluence, the adherent cells were

digested by trypsin and collected in a 1.5 mL centrifuge tube by centrifuging for

1 min under 10,000 rpm. The supernatant was completely removed, followed

by adding 200 µL GA buffer + 4 µL RNaseA (100 mg/mL) and vortex for 15 s.

(2) After incubation at room temperature for 5 min, the resulting solution was

mixed with 20 µL Proteinase K solution, followed by adding 200 µL GB buffer

and incubation in the 70 water both for 10 min. The droplets on tube wall

were removed by centrifugation for 30 s.

(3) The resulting solution was mixed with 200 µL absolute alcohol (floc will occur)

followed by centrifugation for 30 s to remove droplets on tube wall.

(4) An adsorption column CB3 was immersed into the resulting mixture followed

by centrifugation at 12,000 rpm for 30 s. After removing the supernatant, the

adsorption column was placed into the centrifuge tube, followed by adding 500

µL GD buffer. After centrifugation for 30 s at 12,000 rpm, the supernatant was

removed.

(5) 600 µL PW buffer was added to the adsorption column, followed by

centrifugation for 30 s at 12,000 rpm. The supernatant was removed. This step

was repeated once more. The adsorption column was then dried at room

temperature to removed residual PW buffer.

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(6) The adsorption column was placed in a new tube, followed by adding 50 µL TE

buffer in the center position of column. After incubation at room temperature

for 2-5 min, the resulting solution was collected by centrifugation for 2 min at

12,000 rpm.

Genomic DNA extraction of cell lines, tumor tissue and whole blood were

confirmed by 1% agarose gel electrophoresis with Gen Green dye. The bands (about

20,000 bases) of genetic DNA were observed in each lanes of Fig. S4, indicating the

success extraction of genetic DNA from 6 blank tissues (adjacent peritumoral tissues

without p53 mutation), 6 tumor tissues (Fig. S4 A) and 10 whole blood samples (Fig.

S4 B) of esophageal cancer patients.

Fig. S4. (A) The agarose gel electrophoresis verification of genetic DNA extraction from blank

tissues and tumor tissues of esophageal cancer patients. M (DNA ladders), Blank tissues 1-6,

Tumor tissues 1-6. (B) The agarose gel electrophoresis verification of genetic DNA extraction

from whole blood of esophageal cancer patients and the health. M (DNA ladders), Blank 1-2 (the

healthy blood) and patient blood samples 1-10.

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Genetic sequencing of wild-type p53 and mutant p53 codon 273 in tumor tissue,

whole blood of esophageal cancer patients and SW620 cell lines

In this work, the PDDC system was used for detecting wild-type p53 and mutant

p53 codon 273 (which localized in the exon 8 of p53 gene). It is reported that

esophageal cancer patients may suffer from mutation in codon 2731. Therefore, to

verify the feasibility of PDDC system in assaying practical clinical samples, we

applied it in detecting wild-type p53 in 6 tumor tissue samples and 10 whole blood

samples obtained from esophageal cancer patients. The genomic DNA in these

samples were extracted using TIANamp® Genomic DNA Kit according to

abovementioned method. The sequences in genomic DNA that contained wild-type

p53 and mutant p53 codon 273 (ca. 256 bp) were amplified by PCR. The PCR was

performed by using a pair of specific primers (forward primer 1: 5’-

ATGGGACAGGTAGGACCTG-3’; reverse primer 1: 5’-

ATCTGAGGCATAACTGCACC-3’). This pair of primers was defined as “amplified

primer”. The amplification was achieved by thermal cycling for 32 cycles in a total

volume of 20 µL of 1×PCR buffer containing 1 µM forward primer, 1 µM reverse

primer, 1.5 mM MgCl2, 50 mM KCl, 5 U of Tag polymerase, 0.15 µM dNTPs, and

3.4 µg of genomic DNA from tumor tissues/whole blood/SW620 cell lines. Each PCR

cycle was initiated by 2 min of denaturation at 94 ,followed by 15 s at 94 ,15 s

at 55 ,25 s at 72 , and then a final extension at 72 within a time-period of 7

min. The amplified sequences were verified by 2% agarose gel electrophoresis with

0.8% Gen Green dye (Fig. S5). Finally, the PCR products were sequenced by

BioSune Co. Ltd to verify the wild-type p53 and mutant p53 codon 273 (Fig. S6).

As shown in Fig. S5, bright bands could be observed in each lane and the sizes of

these bands were ca. 256 bases, indicating that target gene was successfully amplified

using PCR (amplified primer was used in this issue). Representative

electropherograms of genetic sequencing were shown in Fig. S6, the wild-type p53

codon 273 (CGT) could be found in all PCR amplicons from tumor tissue and whole

blood of esophageal cancer patients. However, we did not find mutations at codon

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273 (CAT) in these samples. It is well known that p53 mutation can only be found in

a small percentage of esophageal carcinoma1. Thus, the clinical samples collected in

this study were p53 mutation-negative samples, which were then employed as the

practical samples for wild-type p53 detection using the developed PDDC system.

Fig. S5. (A) Agarose gel electrophoresis of PCR amplicons containing p53 codon 273 from tumor

tissues and whole blood of esophageal cancer patients. The lanes from left to right: blank tissue 1-

6, tumor tissue 1-6, M (DNA ladders), blank blood 1-2, patient blood 1-10. (B) Agarose gel

electrophoresis of PCR amplicons containing p53 codon 273 from SW620 cell lines. M: DNA

ladders, 1: PCR amplicon.

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Fig. S6. Representative electropherograms of genetic sequencing for p53 gene. (A) Tumor tissue

samples from 1 to 6, (B) Whole blood samples from 1 to 10.

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Detection of p53 gene in practical samples using PDDC system

To detect the target P53 gene in practical samples (cell lines, tumor tissue and

human blood), the asymmetric PCR amplicons of gene segment contained p53 codon

273 were achieved by using another pair of specific primers (forward primer 1: 5’-

GGTAATCTACTGGGACGGAAC-3’; reverse primer 1: 5’-

GGTCTCTCCCAGGACAGG-3’). This pair of primers was defined as “quantitative

primer”. The asymmetric amplification was achieved by thermal cycling for 38 cycles

in a total volume of 20 µL of 1×PCR buffer containing 1 µM forward primer, 0.2 µM

reverse primer, 1.5 mM MgCl2, 50 mM KCl, 5 U of Tag polymerase, 0.15 µM dNTPs,

and 3.4 µg of genomic DNA. Each PCR cycle was initiated by 2 min of denaturation

at 94 , followed by 15 s at 94 ,15 s at 53 ,15 s at 72 , and then a final

extension at 72 within a time-period of 7 min. The amplified products were

verified by 3% agarose gel electrophoresis with Gen Green dye (Fig. S7). Since the

asymmetric PCR products are single-stranded, they were then directly introduced to

the PDDC system and quantified using abovementioned method.

Fig. S7. Confirmation of asymmetric PCR amplicons (quantitative primer was used in this issue)

containing p53 codon 273 from tumor tissues and whole blood of esophageal cancer patients by

agarose gel electrophoresis. The lanes from left to right: blank tissues 1-6, tumor tissues 1-6, M

(DNA ladders), patient blood samples 1-10.

To demonstrate the ability of PDDC system in assaying p53 gene in practical

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clinical samples, we challenged it with the asymmetric PCR amplicons from tumor

tissues and bloods of esophageal cancer patients. The genetic sequencing method (Fig.

S6) have proven that these samples involved no mutant p53 gene. So, theoretically,

the response signals of PDDC system towards these samples were the same when the

amplicons with same OD value were used. As shown in Fig. S8, the relative

fluorescence intensity of PDDC system responding to PCR amplicons (with same OD

value) from different samples were almost the same, indicating that there existed no

mutant p53 codon 273 in these samples [The amplicons from adjacent gastric mucosa

(without mutant p53) were selected as the “blank PCR amplicons”, and the relative

fluorescence intensity was calculated by the following formula: Relative fluorescence

intensity = Responding fluorescence intensity of samples/Responding fluorescence

intensity of blank PCR amplicons × 100%]. This result agreed well with the genetic

sequencing result. Hence, we could make a conclusion that the developed PDDC

system could be preliminary applied for gene diagnosis.

Fi

g. S8. The relative fluorescence intensity of PDDC system towards asymmetric PCR amplicons

from different clinical samples. NS: no significant difference.

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Table S2. Comparison of analytical properties of the developed PDDC strategy with other

methods.

Method LOD Linearity range Category Reference

Pendulum-type optical DNA nanodevice

800 pM 800 pM to 163 nM

Fluorescent assay

2

Fluorescence near gold nanoparticles 100 pM 100 pM to 1 nM Fluorescent assay

3

Silver nanoclusters fluorescent probe 290 pM 5 nM to 100 nM Fluorescent assay

4

Exonuclease Ⅲ-assisted amplification detection of nucleic acid

120 pM 200 pM to 40 nM

Fluorescent assay

5

Ratiometric fluorescent sensing method based on DNA-templated gold nanoclusters

300 pM 1 nM to 10 nM Fluorescent assay 6

Self-assembly of DNA nanoparticles through multiple catalyzed hairpin assembly

7.7 pM 10 to 600 pM Colorimetric method 7

Paper-based electrochemical biosensor 2.3 nM 10 to 200 nM Electrochemical method

8

Paper-based colorimetric DNA sensor using pyrrolidinyl peptide nucleic acid-induced AgNPs aggregation

1.03 nM 20 to 2500 nM Colorimetric method 9

Polymerization-driven DNA dominoes collapse (PDDC) strategy

15 pM 50 pM to 30 nM Fluorescent assay

This work

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Table S3. Recovery tests for target DNA in human serum.

SamplesTarget DNA

added (nM)Target DNA detected (nM) RSD (n=3) Recovery (%)

1 100 101.36 7.42% 101.36

2 40 39.45 1.59% 98.62

3 20 21.61 2.03% 108.05

4 10 9.41 0.57% 94.10

5 5 5.09 1.68% 101.80

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References1 A. G. Casson, T. Mukhopadhyay, K. R. Cleary, J. Y. Ro, B. Levin and J. A. Roth, Cancer Res, 1991, 51, 4495.

2 J. Xu, Z. Wu, H. Li, Z. Wang, J. Le, T. Zheng and L. Jia, Biosens. Bioelectron., 2016, 86, 1024.3 Y. Cheng, T. Stakenborg, P. Van Dorpe, L. Lagae, M. Wang, H. Chen and G. Borghs, Anal. Chem., 2011, 83, 1307.

4 H. Zhao, L. Wang, J. Zhu, H. Wei and W. Jiang, Talanta, 2015, 138, 163.5 W. Yang, J. Tian, Y. Ma, L. Wang, Y. Zhao and S. Zhao, Anal Chim Acta, 2015, 900, 90.6 Z. Li, Y. Wu and W. Tseng, ACS Appl. Mater. Interfaces, 2015, 7, 23708.7 H. He, J. Dai, Y. Meng, Z. Duan, C. Zhou, B. Zheng, J. Du, Y. Guo and D. Xiao, Talanta, 2018, 179, 641.

8 P. Teengam, W. Siangproh, A. Tuantranont, C. S. Henry, T. Vilaivan and O. Chailapakul, Anal Chim Acta, 2017, 952, 32.

9 P. Teengam, W. Siangproh, A. Tuantranont, T. Vilaivan, O. Chailapakul and C. S. Henry, Anal. Chem., 2017, 89, 5428.

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