Quantitative chemical tests for biological molecules

• describe stage 1: testing solutions of known reducing sugar concentration

  • use at least 5 known concentrations of reducing sugars and pure water
  • perform the normal test for reducing sugars (benedicts and heat at 80 degrees)
  • use same volume of each sample and same volume of benedict’s added to each sample
  • must be placed in same water bath at same time
  • must be left for same length of time
  • a range of different colour will be produced
• describe stage 2: filtering

  • each solution must be filtered to remove the brick-red precipitate
  • care must be taken not to tear the filter paper, so no precipitate passes through
  • filtering the precipitate is important as the absorbance readings are determined by the amount of blue benedict’s solution remaining in the filtrate
• describe stage 3: using the colorimeter

  • calibrate the colorimeter to zero using the highest concentration of reducing sugar using either the red or orange filter
  • it sets the absorbance of this reference sample as zero
  • measure the absorbance of the other samples
• how does colorimetry work?

  the colorimeter shine light of red wavelength through the solution - a blue solution will absorb red wavelengths
• what is standard deviation?

  measuring the spread of data around a mean, the bigger the standard deviation the larger the spread of data
• describe stage 4: creating the calibration curve of reducing sugar concentration versus absorbance

  • for each concentration of reducing sugar, since the sample volume is known, the mass of reducing sugar can be calculated
  • plot a graph either: concentration of reducing sugar vs absorbance, mass of reducing sugar vs absorbance
  • draw a line of best fit and use it to find the concentration of the unknown
• how do you do standard deviation?

  • square the differences, to deliberately accentuate the effect of the larger ones (value - mean, square difference)
  • then add values together and divide by n-1 (n = num of values)
  • then square root
• what results are expected for a low glucose concentration?

  • solution remains blue
  • red light absorbed
  • high absorbance reading
  • low transmission reading
• what results are expected for a high glucose concentration?

  • solution colourless/pale blue
  • red light transmitted
  • low absorbance reading
  • high transmission reading
• what is a biosensor?

  a device that takes a biological or chemical variable which cannot easily be measured, and converts it into an electrical signal so it can be measured
• how do biosensors work?

  Molecular recognition - a protein or single strand of DNA (ssDNA) is immobilised to a surface (e.g. a glucose test strip). This will interact with or bind to the specific molecule under investigation

  Transduction - this interaction will cause a change in a transducer (detects changes - e.g. pH and produces a response such as the release of an immobilised due on a test strip or an electric current in a glucose-testing machine)

  Display - produces a visible, qualitative or quantitative signal such as a particular colour on a test strip or reading on a test machine
• what are potential uses of biosensors?

  • determine presence and/ or concentration of biological molecules (e.g. blood glucose concentration or pregnancy tests)
  • detecting contamination in water
  • detecting pathogens and toxins in food
  • detecting airborne bacteria in counter-bioterrorism programmes