Chapter 19: Q20P (page 488)
Explain how signal amplification is achieved in enzyme linked immunosorbent assays.
In enzyme- linked immunosorbent assay (ELISA), enzyme catalyzes the reaction in which colored or fluorescent product is formed. So, many molecules of colored product are created for each analyte molecule.
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The end of Box 19-2 states that โthe advantage of up conversion for biomedical probes is that low energy near-infrared (800 to 1000 nm) incident radiation stimulates little background emission from the complex biological matrix that can be highly fluorescent under visible radiation.โ Suggest why near-infrared radiation stimulates less emission than visible radiation and why this behavior is useful.
The spreadsheet gives the productfor four pure compounds and a mixture at infrared wavelengths. Modify Figure 19-4 to solve four equations and find the concentration of each compound. You can treat the coefficient matrix as if it were molar absorptivity because the path length was constant (but unknown) for all measurements.
The indicator xylenol orange (Table 12-3) forms a complex with Zr(IV)in HCIsolution. Prepare a Job plot from the data in the table and suggest the stoichiometry of the complex (xylenol orange).
The protein bovine serum albumin can bind several molecules of the dye methyl orange. To measure the binding constant K for one dye molecule, solutions were prepared with a fixed concentration of dye and a larger, variable concentration of protein (P). The equilibrium is Reaction 19-18, with X 5 methyl
orange.
Experimental data are shown in cells A16-D20 in the spreadsheet on the next page. The authors report the increase in absorbanceat 490 nm as P is added to X. X and PX absorb visible light, but P does not. Equilibrium expression 19-20 applies and [PX] is given by Equation 19-21. Before P is added, the absorbance is. The increase in absorbance when P is added is
The spreadsheet uses Solver to vary K and in cells B10:B11 to minimize the sum of squares of differences between observed and calculatedin solutions with different amounts of P. Cell E16 computes [PX] from Equation 19-21, which is Equation A on line 6 of the spreadsheet. Cells F16 and G16 find [X] and [P] from mass balances. Cell H16 computes which is Equation B on line 7.
To estimate a value of K in cell B10, suppose that 50% of X has reacted in row 20 of the spreadsheet. The total concentration of X is. If half is reacted, thenThe binding
constant is which we enter as our guess for K in cell B10. We estimatein cell B11 by supposing that 50% of X has reacted in row 20. In Equation B on line 7, .The measured
value ofin row 20 is 0.0291 and we just estimated that. Therefore, our guess for localid="1668328314124" in cell B11 is
Your assignment is to write formulas in columns E through J of the spreadsheet to reproduce what is shown and to find values in cells E17:J20. Then use Solver to find K andin cells B10:B11 to minimizein cell I21.
Spectroscopic data for the indicators thymol blue (TB),semithymol blue (STB), and methylthymol blue (MTB) are shown in the table. A solution ofTB,STB,MTB in a1.000-cm cuvet had absorbances of 0.412at455nm,0.350 at485nm, and 0.632 at545nm. Modify the spreadsheet in Figure 19-4 to handle three simultaneous equations and findandin the mixture.
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