Synthesis · Target-triggered hybridization chain reaction for ultrasensitive dual-signal miRNA detection

6 structured synthesis routes

Completeness describes how fully the route could be reconstructed from the main article and supporting information.

Partial recipeSource: Both

Route 1: Electrochemical

SI p.4-5 · Electrochemical measurements; Optimization of sensor · Fig. S1A

Metal precursors0.1% HAuCl4

SolventsN-PCD solution solvent not specified; HAuCl4 electrolyte solution

Additives0.1 M KCl

Atmospherenot specified

Temperaturedrying temperature not specified

TimeAu electrodeposition 30 s; drying time not specified

Substrate orientationpolished bare glassy carbon electrode

Oxidant / reductantelectrodeposition of Au from HAuCl4 by i-t at -0.2 V

Work-upBare GCE polished; 8 uL N-PCD solution (1 mg/mL) dropped and dried; electrode placed in 0.1% HAuCl4 containing 0.1 M KCl for AuNP electrodeposition.

Show 3 structured reagent records

Role	Reagent	Amount / concentration	Source
Other	N-PCD solution	8 uL · 1 mg/mL	SI p.4-5 · Electrochemical measurements; Optimization of sensor · Fig. S1A
Metal Source	HAuCl4	0.1%	SI p.4-5 · Electrochemical measurements; Optimization of sensor · Fig. S1A
Electrolyte	KCl	0.1 M	SI p.4-5 · Electrochemical measurements; Optimization of sensor · Fig. S1A

Partial recipeSource: SI

Route 2: Other

SI p.4 · Cell culture and RNA extraction

SolventsDMEM medium; RNase-free water

Additives10% fetal bovine serum; 1% penicillin/streptomycin; trypsin; Trizol

Atmosphere5% CO2 for cell culture

Temperature37 deg C cell culture; extraction on ice bath

Timeserum-free medium for 48 h; trypsin 1-2 min

Work-upCells collected by centrifugation; total RNA extracted by Trizol method on ice.

Show 6 structured reagent records

Role	Reagent	Amount / concentration	Source
Other	L02 liver cells	Not specified	SI p.4 · Cell culture and RNA extraction
Other	MCF-7 cells	Not specified	SI p.4 · Cell culture and RNA extraction
Solvent	DMEM medium	Not specified	SI p.4 · Cell culture and RNA extraction
Additive	fetal bovine serum	10%	SI p.4 · Cell culture and RNA extraction
Additive	penicillin/streptomycin	1%	SI p.4 · Cell culture and RNA extraction
Other	Trizol	Not specified	SI p.4 · Cell culture and RNA extraction

Complete recipeSource: Both

Route 3: Other

main p.3 · 2.3 Fabrication of the biosensor · Scheme 1

Metal precursorsAuNPs/N-PCD/GCE electrode

Linker precursorsT4-DNA capture probe; DNA1-Fc; DNA2-MB

SolventsTris-HCl buffer pH 7.4 rinse; RNase-free water wash; 10 mM PBS pH 7.0 for DPV

Additives6-mercapto-1-hexanol (MCH)

Atmospherenitrogen atmosphere for DPV

Temperature4 deg C overnight T4-DNA immobilisation; 37 deg C for MCH, miRNA and HCR incubations

TimeT4-DNA overnight; MCH 0.5 h; miRNA 70 min; HCR/co-incubation 70 min

Substrate orientationAuNPs/N-PCD/GCE

Work-upRinsed with Tris-HCl buffer before each modification step; washed with RNase-free water after miRNA incubation.

ActivationHCR triggered by DNA1-Fc and DNA2-MB mixture

Show 6 structured reagent records

Role	Reagent	Amount / concentration	Source
Other	T4-DNA solution	8 uL · 1 uM	main p.3 · 2.3 Fabrication of the biosensor · Scheme 1
Additive	6-mercapto-1-hexanol (MCH)	4 uL · 1 mM	main p.3 · 2.3 Fabrication of the biosensor · Scheme 1
Other	miRNA-21	8 uL · different concentrations	main p.3 · 2.3 Fabrication of the biosensor · Scheme 1
Other	DNA1-Fc and DNA2-MB mixture	1 uM in main text; SI optimisation text says 1.0 mM	main p.3 · 2.3 Fabrication of the biosensor · Scheme 1
Solvent	Tris-HCl buffer	rinse · pH 7.4	main p.3 · 2.3 Fabrication of the biosensor · Scheme 1
Solvent	RNase-free water	wash	main p.3 · 2.3 Fabrication of the biosensor · Scheme 1

Complete recipeSource: Main

Route 4: Other

main p.2-3 · 2.1 Preparation of ZIF-8 and N-PCD · Scheme 2

Metal precursorsdried ZIF-8 precursor

Linker precursorsZIF-8-derived 2-methylimidazolate

AtmosphereN2 protection during calcining

Temperature30 to 100 deg C within 30 min; then to 800 deg C at 2 deg C min-1; hold at 800 deg C

Time2 h hold at 800 deg C

Substrate orientationquartz boat

Work-upanneal/cool to room temperature after calcination

Activationhigh-temperature carbonisation of dried ZIF-8

Show 2 structured reagent records

Role	Reagent	Amount / concentration	Source
Other	dried ZIF-8	not reported	main p.2-3 · 2.1 Preparation of ZIF-8 and N-PCD · Scheme 2
Other	N2	protective atmosphere	main p.2-3 · 2.1 Preparation of ZIF-8 and N-PCD · Scheme 2

Complete recipeSource: Both

Route 5: Other

SI p.12-13 · Table S1 · Table S1

Linker precursorsDNA precursor strands a21 or a141, b, c and d

Solvents10 mM Tris-HCl buffer

Additives20 mM TCEP; 50 mM MgCl2; pH 8.0

Atmospherenot specified

Temperature95 deg C for annealing start; 4 deg C on ice; stored at -20 deg C

Time10 min at 95 deg C; ice annealing time not specified

Oxidant / reductantTCEP reducing agent

Work-upDNA solutions mixed equally and annealed in a PCR instrument, then on ice

Show 8 structured reagent records

Role	Reagent	Amount / concentration	Source
Other	DNA precursor a21	mixed equally · final concentration 4 uM	SI p.12-13 · Table S1 · Table S1
Other	DNA precursor a141	used for miRNA-141 variant	SI p.12-13 · Table S1 · Table S1
Other	DNA precursor b	mixed equally · final concentration 4 uM	SI p.12-13 · Table S1 · Table S1
Other	DNA precursor c	mixed equally · final concentration 4 uM	SI p.12-13 · Table S1 · Table S1
Other	DNA precursor d	mixed equally · final concentration 4 uM	SI p.12-13 · Table S1 · Table S1
Solvent	Tris-HCl buffer solution	10 mM, pH 8.0	SI p.12-13 · Table S1 · Table S1
Reductant	TCEP	20 mM	SI p.12-13 · Table S1 · Table S1
Additive	MgCl2	50 mM	SI p.12-13 · Table S1 · Table S1

Complete recipeSource: Both

Route 6: Other

main p.2 · 2.1 Preparation of ZIF-8 and N-PCD

Metal precursors0.19 g Zn(CH3COO)2 in 100 mL methanol

Linker precursors0.29 g 2-methylimidazole in 50 mL methanol

Solventsmethanol; absolute ethanol wash

Atmospherenot specified; presumed ambient during solution synthesis

Temperature25 during stirring; 60 during vacuum drying

Time12 h stirring plus 12 h standing; 12 h drying

Work-up2-methylimidazole methanol solution slowly poured into zinc acetate solution; product collected after 3 min centrifugation at 10000 rpm; washed in absolute ethanol 2-3 times.

Activationdried at 60 deg C for 12 h under vacuum

Show 4 structured reagent records

Role	Reagent	Amount / concentration	Source
Metal Source	Zn(CH3COO)2	0.19 g	main p.2 · 2.1 Preparation of ZIF-8 and N-PCD
Linker	2-methylimidazole	0.29 g	main p.2 · 2.1 Preparation of ZIF-8 and N-PCD
Solvent	methanol	100 mL + 50 mL	main p.2 · 2.1 Preparation of ZIF-8 and N-PCD
Solvent	absolute ethanol	washed 2-3 times	main p.2 · 2.1 Preparation of ZIF-8 and N-PCD