The Mechanistic Study on Traditional Chinese Silver Staining and its Application as Phosphate Ion Detection
Mechanism of Chinese Silver Staining Diagnosis and its Application for Phosphate Ion DetectionKings CollegeChu Yat HoYip Tsz FungDavid IuZuo Long LongHo Hiu Fai
Traditional Chinese Silver Staining Technique
(1) INTRODUCTIONWhy does silvers colour change?HypothesisAg undergoes specific reaction with the proteins and amino acids present in the egg white.
ExperimentationTest different amino acids suspectedLeucineMethionineCysteinePhenylalanineAsparagineHistidineAlanineProlineGlutamic acidGlycine
ValineAspartic acid(2) MECHANISMResult: Only cysteine show characteristic color change
Purple and blue(2) MECHANISM
Silvery whiteResult: Only cysteine show characteristic color changepH dependenceConditionColour changeResultAcidicSilver to YellowAlkalineSilver to Blue
Question 2: Why color depends on pH?Hypothesis: Cysteine has different structure under different pH conditions.What is CYSTEINE?
It has unique thiol group, which we proposed is responsible for coloration.pH dependence
Proposed mechanism
DiscussionCarboxylate group is responsible for the yellow complexes in acidic conditionAmine group is responsible for the blue complexes in alkaline conditionBlue complexes are under thermodynamic control
VerificationHypothesis: Cysteamine will give yellow complexes, 3-mercaptopropinoic acid will give blue complexes, irrespective to pH conditions.
Experimentation: use 3-mercaptopropinoic acid and cysteamine to verify the mechanism.
(Carboxyl group removed)(Amine group removed)CysteineCysteamine3-Mercaptopropionic acidpH 4.0 bufferYellowNo observable changeYellowpH 10.0 bufferBlueBlueYellow
Catalytic Effect of anionsExperimentation: to test the catalytic effect of different anions on the coloration on silver plate
Results:PO43- HCO3- Cl-PO43- +Cl- + HCO3-PO43-HCO3-Cl-
Colour of the silver plateControl (Distilled water was added)(Overall conc. of PO43- : 0 M )2.0 cm3 5.00 x 10-6 M PO43- added(Overall conc. of PO43- : 3.125 x 10-7 M )2.0 cm3 5.00 x 10-5 M PO43- added(Overall conc. of PO43- : 3.125 x 10-6 M ) 2.0 cm3 5.00 x 10-4 M PO43- added(Overall conc. of PO43- : 3.125 x 10-5 M )
DiscussionPhosphate ion can significantly promote the coloration in both acidic and alkaline buffer solution.
The coloration speeds up from 1 hr to 10 min under the presence of phosphate ion.Screening test for Chronic Renal FailureChronic Renal Failure patients exhibit proteinuriaProtein concentration in urine >0.43 g/dm3High level of proteins & amino acids discharged in urine Measurement of protein concentration = test for proteinuriaScreening test for Chronic Renal FailureSuggested procedureCollect first morning urine samples from test subjectsRefrigerated at 4oC for