All chemical reagents are analytical-graded reagents. Hydrogen tetrachloroaurate trihydrate (HAuCl4), Phosphate buffered saline (PBS buffer), trisodium citrate solution (C6H5Na3O7, ≥99%), (3-Mercaptopropyl)methyldimethoxysilane (MPDMS, >95%), cystamine dihydrochloride (cystamine), dextran 70 (MW ≈ 70,000), sodium periodate (NaIO4), 11-mercaptoundecanoic acid (MUA; ≥95%), 6-mercapto-1-hexanol (MCH; ≥97%), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), ethanolamine (EA), mouse IgG, streptavidin, monoclonal anti-HMGB1 antibody, and high mobility group box 1 (HMGB1) protein were from Sigma-Aldrich (St. Louis, MO, USA). Sulfuric acid (H2SO4; ≥98%), hydrogen peroxide (H2O2), ethanol, and acetate buffer were purchased from Fluka (Buchs, Switzerland). Ultrapure deionized water (18.2 MΩ·cm−1, Milli-Q pure water purification system, Millipore Ltd., Burlington, MA, USA) was used for preparing solutions. The optical fiber probe was multimode plastic-clad silica optical fiber (model F-MBC, Newport), with core and cladding diameters of 400 and 430 μm, respectively, bought from Instant NanoBiosensors Co., Ltd. (Taipei, Taiwan). Sensing chips (poly (methyl methacrylate) (PMMA) plates) were prepared using a CO2 laser engraving machine (New Taipei, Taiwan).
Trisodium citrate solution
Manufactured by Merck Group
Sourced in United States
Trisodium citrate solution is a laboratory reagent that is commonly used as an anticoagulant. It functions by chelating calcium ions, which are necessary for the blood clotting process. The solution is typically used in various medical and scientific applications that require the prevention of blood clot formation.
Automatically generated - may contain errors
Lab products found in correlation
Cystamine dihydrochloride, by Merck Group (2 mentions)
Streptavidin, by Merck Group (2 mentions)
Dextran 70, by Merck Group (1 mentions)
11 mercaptoundecanoic acid, by Merck Group (1 mentions)
3 mercaptopropyl methyldimethoxysilane, by Merck Group (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride edc, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
6 mercapto 1 hexanol, by Merck Group (1 mentions)
Hydrogen tetrachloroaurate trihydrate, by Merck Group (1 mentions)
Mouse igg, by Merck Group (1 mentions)
Hydrogen tetrachloroaurate, by Merck Group (1 mentions)
1 ethyl 3 3 dimethyl aminopropyl carbodiimine hydrochloride, by Merck Group (1 mentions)
Human serum albumin has, by Merck Group (1 mentions)
Goat anti mouse igg antibody, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Ethanolamine, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
N hydroxysuccinimide, by Merck Group (1 mentions)
Potassium permanganate, by Merck Group (1 mentions)
Chloroauric acid solution, by Merck Group (1 mentions)
5 protocols using trisodium citrate solution
1
Plasmonic Biosensor Platform for HMGB1 Detection
2
Functionalized Graphene-based Biosensor
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Hydrogen tetrachloroaurate (III) (HAuCl4·4H2O, ≥99.9% trace metals basis), trisodium citrate solution (C6H5Na3O7, ≥99%), cystamine dihydrochloride (cystamine), N-hydroxy-succinimide (NHS), 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimine hydrochloride (EDC), (3-mercaptopropyl)-trimethoxysilane (MPTMS, >95%), graphite flakes (99% carbon basis), hydrochloric acid, potassium permanganate (KMnO4; ≥99%), glycine, ethanolamine (EA), human serum albumin (HAS), goat anti-mouse IgG antibody, mouse IgG (isoelectric point (pI) = 7), high mobility group box 1 (HMGB1), N-(+)-biotinyl-3-aminopropylammonium trifluoroacetate (Biotin-NH2), and streptavidin were bought from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen peroxide (H2O2) and sulfuric acid (H2SO4; ≥98%) were bought from Fluka (Buchs, Switzerland). Ethanol and acetone were bought from HyBiocareChem (New York, NY, USA). Sodium citrate was bought from J.T. Baker (Phillipsburg, NJ, USA). All aqueous solutions in this study were prepared using the Milli-Q pure water purification system of U.S. Millipore Ltd. (Burlington, MA, USA) (resistivity = 18.2 MΩ/cm). All biological samples were prepared using phosphate-buffered saline (PBS) having a pH of 7.4.
3
Synthesis of Monodisperse Gold Nanoparticles
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Gold NPs were synthesized following Frens protocol. [36] Briefly, 2 ml of a 0.5 wt% chloroauric acid solution (Sigma-Aldrich) was diluted in 96 ml water and brought to a boil under reflux. Next, 2 ml of trisodium citrate solution (Sigma-Aldrich) was swiftly injected to the solution with vigorous stirring. Upon addition of the trisodium citrate, the color of the solution changed to blue and then gradually became red. This color change is attributed to the increase in size of gold NPs as the citrate ions reduce gold (III). [36] This procedure produces highly spherical monodisperse gold NPs. [37] After cooling the solution in the room temperature, the NPs were PEGylated by mixing 1 ml of a 1 wt% methoxy-PEG-thiol (5 kDa, JenKem Technology) with the NP solution for 8 hrs.
The diameter (d = 41 nm) and polydispersity (PDI= 0.17) of the PEGylated gold NPs were measured using dynamic light scattering (Malvern ZetaSizer Nano-ZS). The diameter of the hard core of these NPs (without PEG) was 23 nm.
The diameter (d = 41 nm) and polydispersity (PDI= 0.17) of the PEGylated gold NPs were measured using dynamic light scattering (Malvern ZetaSizer Nano-ZS). The diameter of the hard core of these NPs (without PEG) was 23 nm.
4
Colloidal Gold Conjugation with Antibody
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Colloidal gold particles (40 nm mean diameter) were prepared according to a previously described procedure [8 ]. Briefly, 100 mL of a 0.01% HAuCl4 solution (Sigma-Aldrich) in a 250 mL conical beaker was boiled thoroughly, and 1.8 mL of 1% trisodium citrate solution (Sigma-Aldrich) was then added with constant stirring. After the color of the solution changed to wine-red after about 45 sec, the solution was boiled for another 5 min. The heat source was removed and the colloidal gold solution was stirred for another 10 min. The solution was stored in a dark bottle at 4℃ and used to prepare the colloidal gold-mAb conjugate as soon as possible. Anti-p27 mAb 8G7D1 was conjugated with the 40-nm colloidal gold particles as previously described [5 (link),6 (link),37 (link)]. The mAb 8G7D1 was subsequently dialyzed with 1 mM Borax (pH 9.0) (Sigma-Aldrich) for 5 h at 4℃. The pH of the gold solution was adjusted to 9.0 with 0.2 M K2CO3 and mixed with 1 mg/mL of mAb. The mixture was then incubated for 30 min at room temperature. The gold conjugate was blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich) for 30 min under at room temperature. The conjugate was then washed three times with PBS containing 1% BSA. The OD of the mAb-gold conjugate was measured at 540 nm by spectrophotometer.
5
Synthesis and SERS Analysis of AgNPs for NOD Detection
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For the SERS analysis of NOD, AgNPs were prepared according to the classical, citrate-reduced procedure, as previously described [83 (link)]. In brief, 100 mL of aqueous solution containing 17 mg of AgNO3 salt (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) was heated to boiling (100 °C) and then 2 mL of 1% trisodium citrate solution (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) was added in drops, with constant stirring. The mixture was boiled for 45 min and left to cool down at room conditions. The freshly prepared colloidal AgNP solution was milky-grey in color, and exhibited an UV-VIS-absorption maximum at 424 nm and a featureless Raman spectrum. As such, prepared colloidal AgNPs according to this method have a size distribution centered around 40 nm [83 (link)]. The preparation of the SERS stock solutions follows the same steps—20 µL of different concentrations ranging from 10−3 to 10−7 M of NOD previously dissolved in ethanol was immersed in 400 µL AgNPs starting from a concentration of 10−5 M down to 10−8 M. For SERS analysis, 60 s exposure time and 5 acquisition at 10% laser power were set for each spectral acquisition.
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