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Multiplex Detection of Cardiac BiomarkersMukesh Digambar Sonawane, Satish Balasaheb Nimse, Keum-Soo Song, Taisun Kim*

Institute for Applied Chemistry and Department of Chemistry, Hallym University, Chuncheon, 200-702, Korea; E-mail: tskim@hallym.ac.kr

Supporting Information

1. Materials S2

2. Instruments S2

3. Composition of used solutions S2

4. Abbreviations S3

5. Probes and target oligonucleotides S4

6. Labeling of antibodies and preparation of Protein-DNA conjugates

6a. Labelling of detection antibodies with Cy5 dye S4

6b. Synthesis and purification of the antibody-DNA conjugates S5

7. Immobilization of cAb-DNA on 9GDNA Chip S6

8. Preparation of a hybridization buffer containing biomarkers and Cy5-dAbs S7

9. Result:

a) Chip result for singleplex detection S8

b) Chip result for multiplex detection S9

c) Cross reactivity study S10

d) Standard graph for multiplex detection S10

e) Chip result for multiplex detection of serum samples S11

10. Reference S11

Electronic Supplementary Material (ESI) for Analytical Methods.This journal is © The Royal Society of Chemistry 2017

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1. Materials

All chemicals were purchased from Sigma-Aldrich Chemicals, Korea. All the oligonucleotides were purchased from Bioneer, Korea. For labeling of the oligonucleotides and antibodies with Cy5, the Cy5Dye mono-reactive NHS ester (PA25001) was purchased from GE Healthcare UK Limited, Buckinghamshire, UK. Cardiac troponin I (cTnI) monoclonal capture antibody (Catalog #. 4T21-560), cTnI monoclonal secondary antibody (Catalog #. 4T21-19C7), N-terminal of the prohormone brain natriuretic peptide (NTproBNP) monoclonal capture antibody (Catalog #. 4NT1-15F11), NTproBNP monoclonal secondary antibody (Catalog #. 4NT1-24E11), Cardiac troponin T (cTnT) monoclonal capture antibody (Catalog #. 4T19-1C11), cTnT monoclonal secondary antibody (Catalog #. 4T19-7E7), were purchased from the HyTest. cTnI, NTproBNP and cTnT antigen were purchased from BioRad as a human serum based liquid i.e. Liquichek cardiac markers plus control (Also contains other antigens including BNP, total CK-MB, hs-CRP, digitoxin and myoglobin).

Glass slides (2.5x7.5 cm) were purchased from Paul Marienfeld GmbH & Co. KG, Germany. All washing solvents for the substrates are of HPLC grade from SK Chemicals, Korea. Ultrapure water (18 M Ω/cm) was obtained from a Milli-Q purification system (Millipore).

Clinical samples (n= 5 in this study included 2 women and 3 men who attended the Fuwai hospital, Beijing, China, during 17th to 20th of January 2017 for whom cTnT was requested as a part of routine health check-up. Clinical investigation has been conducted according to the principles expressed in the Declaration of Helsinki. Ethical approval was given by the medical ethics committee of Fuwai hospital, Beijing, China, with the reference number of 2016-ZX40 which was approved on 29th August, 2016. Collection of human plasma from five individuals including 2 women and three men for quantification of serum troponin T has been approved by this committee. Furthermore, the informed consent was obtained from these individuals.

2. Instruments

Oligonucleotides were spotted using Qarray2 microarrayer (Genetix Technologies, Inc.) Hybridization was done at 250C using the commercial incubator and then the slides were dried using the commercial centrifuge (1000rpm). The fluorescence signal of the microarray was measured on ScanArrayLite (GSI Lumonics), and the images were analyzed by Quant Array software (Packard Bioscience).

3. Composition of used solutions:

1. Dilution Buffer: 0.2%milk, 0.1% NaN3, 5mM EDTA, 1XPBS

2. Hybridization buffer solution: 5%FA, 5%butanol, 8XSSC

3. Washing buffer solution: 4XSSC

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4. Abbreviations:

cTnI: Cardiac Troponin I

NTproBNP: N-terminal pro b-type natriuretic peptide

cTnT: Cardiac Troponin T

cTnI-cAb: Cardiac Troponin I monoclonal capture antibody

NTproBNP-cAb: N-terminal pro b-type natriuretic peptide conjugated with monoclonal capture antibody

cTnT-cAb: Cardiac Troponin T monoclonal capture antibody

cTnI-dAb: Cardiac Troponin I monoclonal detection antibody

NTproBNP-dAb: N-terminal pro b-type natriuretic peptide monoclonal detection antibody

cTnTdAb: Cardiac Troponin T monoclonal detection antibody

cTnIcAb-DNA: Cardiac Troponin I monoclonal capture antibody conjugated to DNA

NTproBNPcAb-DNA: N-terminal pro b-type natriuretic peptide monoclonal capture antibody conjugated to DNA

cTnTcAb-DNA: Cardiac Troponin T monoclonal capture antibody conjugated to DNA

Cy5-cTnI-dAb: Cardiac Troponin I monoclonal detection antibody conjugated to Cy5

Cy5-NTproBNP-dAb: N-terminal pro b-type natriuretic peptide monoclonal detection antibody conjugated to Cy5

Cy5-cTnT-dAb: Cardiac Troponin T monoclonal detection antibody conjugated to Cy5

Probe-1: 9G probe complimentary to DNA1

Probe-2: 9G probe complimentary to DNA2

Probe-3: 9G probe complimentary to DNA3

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5. Probes and target oligonucleotides:

Table S1. Sequences and the nomenclature of probes and target oligonucleotides

Probe/DNA Antigen SequenceProbe-1 cTnI 5’-GGG GGG GGG CTT TAT CAT TCG CTA TGG CCT GCC CG-3’Probe-2 NT-proBNP 5’-GGG GGG GGG CTT TAT CAT ACG ACT TGG GGA GGC GC-3’Probe-3 cTnT 5’-GGG GGG GGG CTT TAT CAT ATA CCT TGC GAG CGC GG-3’DNA1 cTnI 5’-NH2-TTT TTT TTT CGG GCA GGC CAT AGC GA-3’DNA2 NT-proBNP 5’-NH2-TTT TTT TTT GCG CCT CCC CAA GTC GT-3’DNA3 cTnT 5’-NH2-TTT TTT TTT CCG CGC TCG CAA GGT AT-3’The GGG GGG GGG sequence in the oligonucleotide probes is used for the immobilization of the oligonucleotide on the 9G DNAChip. Whereas the CTT TAT CAT in the oligonucleotide probe is used as a vertical spacer. The TTT TTT TTT sequence in the target oligonucleotides are also used as a vertical spacer for cAb-DNA.

6. Labelling of antibodies and preparation of antibody-DNA conjugates

6a. Labelling of detection antibodies with Cy5 dye

Figure S1: Nanodrop data depicting the ratio of Cy5 dye molecules conjugated to each dAb, A) Cy5-cTnI-dAb (3.21:1), B) Cy5-NT-proBNP-dAb (3.03:1), C) Cy5-cTnT-dAb (2.56:1).

Briefly, the Cy5-cTnI-dAb, Cy5-NT-proBNP-dAb and Cy5-cTnT-dAb were obtained by the reaction of the amine function of cTnIdAb, NTproBNPdAb and cTnTdAb with the Cy5-Dye mono-reactive NHS ester by following the standard protocol provided by the manufacture with the mono-reactive Cy5DyeTM (GE Healthcare UK Limited, Buckinghamshire, UK).

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6b. Synthesis and purification of the antibody-DNA conjugates

Figure S2: Nanodrop data depicting the ratio of DNAs conjugated to each cAb, A) cTnI-cAb-DNA1 (1:1), B) NT-proBNP-cAb-DNA2 (1:1), C) cTnT-cAb-DNA3 (1:1).

The cTnI-cAb-DNA1, NT-proBNP-cAb-DNA2, cTnT-cAb-DNA3 were synthesized by the reaction of thioalated cTnI antibody, thiolated NTproBNP antibody and thiolated cTnT antibody with the sulfo-SMCC activated DNA1, DNA2, DNA3 respectively following the reported method [1].

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7. Immobilization of cAb-DNA on 9GDNA Chip

Table S2: Preparation of cAb-DNA solution for immobilization of cAb-DNAs on 9GDNA Chip for singleplex and multiplex detection

cAb-DNA Concentration Singleplex Multiplex

cTnI NTproBNP cTnT

cTnIcAb-DNA1 25µg/ml 5µL - - 5µL

NTproBNPcAb-DNA2 25µg/ml - 5µL - 5µL

cTnTcAb-DNA3 25µg/ml - - 5µL 5µL

Dilution Buffer - 25µL 25µL 25µL 15µL

Hybridization Buffer - 30µL 30µL 30µL 30µL

Singleplex detection:

As shown in Table S2, each hybridization chamber of 9G DNAChip was covered with the 60µL mixture of 5µL of individual cTnIcAb-DNA (25µg/ml) or NTproBNPcAb-DNA (25µg/ml) or cTnTcAb-DNA (25µg/ml), 25µL of dilution buffer and 30µL of hybridization buffer. The mixture was allowed to immobilize at 250C for 4 Hr. The 9G DNAChip was rinsed twice for 2 min each with washing buffer in order to remove the excess cAb-DNA and dried by using commercial centrifuge (1000 rpm).

Multiplex detection:

As shown in Table S2, each hybridization chamber of 9G DNAChip was covered with the 60µL mixture of 5µL of cTnIcAb-DNA1 (25µg/ml), NTproBNPcAb-DNA2 (25µg/ml), cTnTcAb-DNA3 (25µg/ml), 15µL of dilution buffer and 30µL of hybridization buffer. The mixture was allowed to immobilize at 250C for 4 Hr. The 9G DNAChip was rinsed twice for 2 min each with washing buffer in order to remove the excess cAb-DNA and dried by using commercial centrifuge (1000 rpm).

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8. Preparation of a hybridization solution containing biomarkers and Cy5-dAbs

Table S3: Preparation of hybridization solution containing biomarkers and Cy5-dAbs singleplex and multiplex detection

Singleplex MultiplexCy5-dAb Conjugate Concentration

cTnI NTproBNP cTnT

Cy5-cTnI-dAb 20µg/mL 5µL 5µL

Cy5-NTproBNP-dAb 20µg/mL 5µL 5µL

Cy5-cTnT-dAb 20µg/mL 5µL 5µL

Liquichek cardiac markers plus control Dilutions Dilution Buffer 95µL 95µL 95µL 85µL

Singleplex:

As shown in Table S3, the biomolecular complexes (Cy5-cTnI-dAb-cTnI-cTnIcAb-DNA1/Cy5-NT-proBNP-dAb-NTproBNP-NTproBNPcAb-DNA2/Cy5-cTnT-dAb-cTnT-cTnTcAb-DNA3 was obtained by loading the mixture obtained by mixing 5μl of the Cy5-cTnI-dAb (20μg/ml)/ Cy5-NT-proBNP-dAb (20μg/ml)/ Cy5-cTnT-dAb (20μg/ml) and 95μl of the antigen solution containing cTnI, NTproBNP, and cTnT on the 9GDNA chip for 4Hr and15 Hr. The excess of antigens and secondary antibodies were removed by washing twice (2min each) with washing buffer and dried in commercial centrifuge (1000 rpm). The fluorescence signal of the microarray was measured on ScanArrayLite, and the images were analyzed by Quant Array software.

Multiplex:

As shown in Table S3, the biomolecular complexes (Cy5-cTnI-dAb-cTnI-cTnIcAb-DNA1, Cy5-NT-proBNP-dAb-NTproBNP-NTproBNPcAb-DNA2 and Cy5-cTnT-dAb-cTnT-cTnTcAb-DNA3 were obtained by loading the mixture obtained by mixing 5μl each of the Cy5-cTnI-dAb (20μg/ml), Cy5-NT-proBNP-dAb (20μg/ml), Cy5-cTnT-dAb (20μg/ml) and 85μl of the antigen solution containing cTnI, NTproBNP, and cTnT on the 9GDNA chip for 4Hr and 15 Hr. The excess of antigens and secondary antibodies were removed by washing twice (2min each) with washing buffer and dried in commercial centrifuge (1000 rpm). The fluorescence signal of the microarray was measured on ScanArrayLite, and the images were analyzed by Quant Array software.

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9. Result:

a) Chip Result for Singleplex Detection

Figure S3: Singleplex detection of cardiac biomarkers at various concentrations

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b) Chip Result for Multiplex Detection

Figure S4: Multiplex detection of cardiac biomarkers at various concentrations

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c) Cross reactivity study

Figure S5: Cross reactivity study for detection of A) cTnI, B) NT-proBNP, C) cTnT using a mixture containing 1 ng/mL of each antigen

d) Standard graph for multiplex Detection

Figure S6: Standard graphs for multiplex detection of A) cTnI, B) NT-proBNP, C) cTnT.

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e) Chip Result for multiplex Detection of serum samples

Figure S7: Multiplex detection of serum sample

10. References:

[1] Y. Jung, J. M. Lee, H. Jung, B. H. Chung, Anal. Chem. 2007, 79, 6534-6541.