Synthesis · Novel 2D CuFe-MOF-based immunoprobe: Addressing antifouling electrochemical immunosensing inadequate sensitivity challenge

8 structured synthesis routes

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

Complete recipeSource: SI

Route 1: Other

1.3 Experimental

Metal precursorsHAuCl4.3H2O

Solventswater

Additivessodium citrate

Atmosphereambient air

Temperatureboiling then ambient

Time0.25 h after colour stabilisation

Oxidant / reductantsodium citrate reductant/stabiliser

Work-upcooled to ambient temperature; stored at 4 C

Scalability contextBatch used 100 mL HAuCl4 solution.

Show 2 structured reagent records

Role	Reagent	Amount / concentration	Source
Metal Source	hydrogen tetrachloroaurate(III) trihydrate (HAuCl4.3H2O)	100 mL · 0.01%	1.3 Experimental
Reductant	sodium citrate	4 mL · 1%	1.3 Experimental

Partial recipeSource: Both

Route 2: Other

2 · 2.3 Preparation of antifouling hydrogel substrate

Metal precursorsAu nanoparticles

Linker precursorsST and HA

SolventsAu NP aqueous dispersion

AdditivesEDC, NHS, PEI, thionine

Atmosphereambient air

Temperatureambient; 4 C gel compositing

Time0.167 h activation; 0.25 h PEI crosslinking; 3 h at 4 C with thionine

Work-upallowed to stand at 4 C for 3 h to composite the gel

ActivationHA carboxy groups activated by EDC/NHS

Show 7 structured reagent records

Role	Reagent	Amount / concentration	Source
Linker	5-serotonin hydrochloride (ST)	0.003 mmol	2 · 2.3 Preparation of antifouling hydrogel substrate
Linker	hyaluronic acid (HA)	0.003 mmol	2 · 2.3 Preparation of antifouling hydrogel substrate
Other	Au NPs (20 nm) dispersion	3 mL	2 · 2.3 Preparation of antifouling hydrogel substrate
Additive	EDC	1 mol	2 · 2.3 Preparation of antifouling hydrogel substrate
Additive	NHS	0.2 mol	2 · 2.3 Preparation of antifouling hydrogel substrate
Additive	PEI	1 mL · 1%	2 · 2.3 Preparation of antifouling hydrogel substrate
Other	thionine (Thi)	1 mg mL-1	2 · 2.3 Preparation of antifouling hydrogel substrate

Complete recipeSource: Main

Route 3: Other

2 · 2.4 Preparation of immunoprobe

Metal precursorsCuFe-MOF

Linker precursorsCuFe-MOF BDC linker; antibody/BSA biomolecules

SolventsPBS pH 7.4; deionized water

AdditivesEDC, NHS, Ab2, BSA

Temperature37; 4 storage

Time0.667 h activation; 2 h Ab2 labelling; 2 h BSA blocking

Work-upcentrifuged and washed with deionized water after activation and labelling; final dispersion in 7.8 mL PBS stored at 4 C

ActivationEDC 0.4 M and NHS 0.1 M in PBS at 37 C for 40 min

Show 5 structured reagent records

Role	Reagent	Amount / concentration	Source
Other	CuFe MOF	0.0078 g	2 · 2.4 Preparation of immunoprobe
Additive	EDC	in 5 mL PBS · 0.4 M	2 · 2.4 Preparation of immunoprobe
Additive	NHS	in 5 mL PBS · 0.1 M	2 · 2.4 Preparation of immunoprobe
Other	labeling antibody (Ab2)	1 mL plus 3 mL PBS · 200 ug mL-1	2 · 2.4 Preparation of immunoprobe
Other	BSA	1 mL · 1%	2 · 2.4 Preparation of immunoprobe

Vague recipeSource: Main

Route 4: Unknown

2 · 2.4 Preparation of immunoprobe

Metal precursorsCu source not specified in this paper

Complete recipeSource: SI

Route 5: Solvothermal

1.3 Experimental

Metal precursorsCu(NO3)2.3H2O

Linker precursorsH2BDC

SolventsDMF

Temperature110

Time36

Work-upcentrifuged, washed twice with ethanol/DMF, dried at 60 C overnight

Activationdried at 60 C overnight

Show 3 structured reagent records

Role	Reagent	Amount / concentration	Source
Metal Source	Cu(NO3)2.3H2O	7.5 mmol	1.3 Experimental
Linker	H2BDC	7.5 mmol	1.3 Experimental
Solvent	DMF	150 ml	1.3 Experimental

Partial recipeSource: Main

Route 6: Other

2 · 2.4 Preparation of immunoprobe

Metal precursorsCu2O dispersion in ethanol

Linker precursorsH2BDC

Solventsethanol and DMF equal volume ratio; methanol for redispersion

Atmosphereambient air

Temperatureambient

Time4

Oxidant / reductantdissolved oxygen oxidises Cu(I) during Cu-BDC formation

Work-upwashed three times by centrifugation at 12000 rpm for 8 min with deionized water/methanol; re-dispersed in 30 mL methanol

Show 4 structured reagent records

Role	Reagent	Amount / concentration	Source
Metal Source	Cu2O	0.05 mmol	2 · 2.4 Preparation of immunoprobe
Linker	H2BDC	0.5 mmol	2 · 2.4 Preparation of immunoprobe
Solvent	ethanol	10 mL Cu2O dispersion in ethanol; equal volume ratio mixture with DMF for linker solution	2 · 2.4 Preparation of immunoprobe
Solvent	DMF	equal volume ratio with ethanol	2 · 2.4 Preparation of immunoprobe

Complete recipeSource: Main

Route 7: Other

2 · 2.4 Preparation of immunoprobe

Metal precursorsCu-BDC nanosheet methanol dispersion; FeCl3.6H2O

Linker precursorsBDC already present in Cu-BDC nanosheets

Solventsmethanol

Atmosphereambient air

Temperatureroom temperature; 60 C drying

Time3 h stirring; overnight drying

Oxidant / reductantFe(III) cation substitution/partial Cu(II) exchange

Work-upwashed with deionized water/ethanol by centrifugation at 10000 rpm for 10 min three times; dried in vacuum at 60 C overnight

Activationvacuum drying at 60 C overnight

Show 3 structured reagent records

Role	Reagent	Amount / concentration	Source
Metal Source	Cu-BDC nanosheet methanol dispersion	30 mL methanol dispersion prepared above	2 · 2.4 Preparation of immunoprobe
Metal Source	FeCl3.6H2O	0.05 mmol	2 · 2.4 Preparation of immunoprobe
Solvent	methanol	30 mL dispersion	2 · 2.4 Preparation of immunoprobe

Complete recipeSource: Main

Route 8: Drop Cast

2 · 2.5 Fabrication of immunosensor

Solventsdeionized water; PBS

AdditivesAu-Thi@ST-HA hydrogel, Ab1, BSA, SCCA, immunoprobe

Atmosphereambient air

Temperature37 for modification/incubation; room temperature for H2O2 reaction

Time0.333 h gel exposure; 0.5 h Ab1; 0.5 h BSA; 0.5 h antigen; 0.5 h immunoprobe; 0.25 h H2O2 reaction

Substrate orientationglassy carbon electrode surface

Oxidant / reductantH2O2 during assay

Work-upwashed with deionized water after each modification step

ActivationGCE polished with 1 um and 0.05 um alumina, sonicated 30 s and rinsed

Show 5 structured reagent records

Role	Reagent	Amount / concentration	Source
Other	antifouling hydrogel	Not specified	2 · 2.5 Fabrication of immunosensor
Other	coating antibody (Ab1)	10 uL · 200 ug mL-1	2 · 2.5 Fabrication of immunosensor
Other	BSA	10 uL · 1%	2 · 2.5 Fabrication of immunosensor
Other	SCCA sample	10 uL	2 · 2.5 Fabrication of immunosensor
Oxidant	hydrogen peroxide	10 uL · 5 mM	2 · 2.5 Fabrication of immunosensor