H64SEN BioSensing

University of Nottingham H64SEN BioSensing Note


Lecture 1



Accuracy, measurement vs True Value, use average to resolve

Precision, (range/standard Dev), use calibration to offset, more important

Limit of Detection (LoD) \(\frac{3 \sigma}{m}\)

Limit of Quantification (LoQ) \(\frac{10 \sigma}{m}\)

\( \sigma = \frac{\sqrt{(x_i – m)^2 }}{ n – 1 } \)

Repeatability, reversibility, reproducibility 

Reversibility: abilityto go back to the base line

Repeatability: get same response in short period of time 

Reproducibility : get same response over a long period of time 


Lecture 2

History of Biosensors

Biosensor classification

  •  Measurand
    • Physical: temperature, pressure;
    • Chemical: concentration, composition;
    • Bio: bacteria, antigens, binding

Biosensor classification by biorecepteor

Antibody/antigen Interactions

  • An immunosensor utilizes the very specific binding affinity of antibodies for a specific compound or antigen. The specific nature of the antibody antigen interaction is analogous to a lock and key fit in that the antigen will only bind to the antibody if it has the correct conformation.
  • The antibody binding capacity is strongly dependent on assay conditions (e.g. pH and temperature)
  •  The antibody-antigen interaction is generally irreversible.

Enzymatic Interactions

  •  the enzyme converting the analyte into a product that is sensor-detectable,
  • detecting enzyme inhibition or activation by the analyte,
  • monitoring modification of enzyme properties resulting from interaction with the analyte.
  • The main reasons for the common use of enzymes in biosensors are:
  •  ability to catalyze a large number of reactions;
  • potential to detect a group of analytes (substrates, products, inhibitors, and modulators of the catalytic activity);
  • suitability with several different transduction methods for detecting the analyte.
  • Notably, since enzymes are not consumed in reactions, the biosensor can easily be used continuously. The catalytic activity of enzymes also allows lower limits of detection compared to common binding techniques. However, the sensor’s lifetime is limited by the stability of the enzyme.

Nucleic acid Interactions

  • genosensors.
  • The recognition process is based on the principle of complementary base pairing, adenine:thymine and cytosine:guanine in DNA
  • If the target nucleic acid sequence is known, complementary sequences can be synthesized, labelled, and then immobilized on the sensor.


Often used in bioreceptors because they are sensitive to surrounding environment and they can respond to all kinds of stimulants. Cells tend to attach to the surface so they can be easily immobilized. Compared to organelles they remain active for longer period and the reproducibility makes them reusable. They are commonly used to detect global parameter like stress condition, toxicity and organic derivatives. They can also be used to monitor the treatment effect of drugs.

Receptor deposition

Spin coating 

  • Important parameters: Viscosity, Spin rate, and Volume

Dip coating

  • Immersion: The substrate is immersed in the solution of the coating material at a constant speed (preferably jitter-free).
  • Start-up: The substrate has remained inside the solution for a while and is starting to be pulled up.
  • Deposition: The thin layer deposits itself on the substrate while it is pulled up. The withdrawing is carried out at a constant speed to avoid any jitters. The speed determines the thickness of the coating (faster withdrawal gives thicker coating material).
  • Drainage: Excess liquid will drain from the surface.
  • Evaporation: The solvent evaporates from the liquid, forming the thin layer. For volatile solvents, such as alcohols, evaporation starts already during the deposition & drainage steps.’


  • Ejection of the material by ions (plasma) from the target with consecutive adsorption (deposition) of this material onto substrate (transducer in our case)
  • Printing (ink jet, cantilever)
  • Electrostatic self assembly or Layer-by-layer
  • Molecular imprinting

Sol-gel technique (entrapment)

  • .Self assembly (monomer template interaction
  • Polymerisation
  • Template extraction

Langmuir-Blodgett deposition

  • Spreading of an amphiphile solution at the air/water interface – all molecules orient with their hydrophilic heads towards the water and hydrophobic tails towards the air
  • Compressing of the monolayer after solvent evaporation to a desired surface pressure (change of the surface tension caused by the presence of the monolayer)
  • The monolayer undergoes different phase transitions
  • Transfer of the Langmuir monolayer at a solid substrate by vertical dipping – Langmuir-Blodgett film

Langmuir-Blodgett deposition

Sample, Surface, Structure  characterisation

Scanning Electron Microscope: SEM

  • a focused beam of high energy electrons across a sample surface.
  • The beam specimen interaction produces a number of signals including;
  • secondary electrons, back scattered electrons and X-rays.
  • The location and intensity of the illumination of the screen depends on the properties of the secondary electrons, and contrast of the real time image reflects the structure of the surface of the object
  • high resolution

Transmission Electron Microscope:TEM

detector below the sample

cant used on thick sample

Atomic Force Microsocpe: AFM

Atomic Force Microscope


Chemical interaction
p-p interaction
Van der Waals forces
Functional groups

Lecture 3: Binding and kinetics, Optical Biosensors

Binding equation derivation

Binding and kinetics

\(K_B = \frac{k_{on}}{k_{off}}\)

Determining \(K_B\)

y=(K_B  [A])/(1+K_B [A])

  • Determine the concentration of ligand at which we get the half maximal concentration of [AB].
  • Plot [A]/y versus [A] we should get a straight line and our intercept on the y-axis
  • Inverse the equation. Intercept on the y-axis (i.e. [A]=0) is 1/KB.

Co-operativity, present of one binding event can help or hinder the next.

Optical Biosensors

Basic principles of light

  • Absorption
  • Scattering (Reflection/refraction)
  • Fluorescence (emission)

Light scattering

When light is scattered from an atom or molecule, most photons are elastically scattered (Rayleigh scattering), such that the scattered photons have the same energy (frequency) and wavelength as the incident photons. However, a small fraction of the scattered light (approximately 1 in 10 million photons) is scattered by an excitation, with the scattered photons having a frequency different from, and usually lower than, the frequency of the incident photons. In a gas, Raman scattering can occur with a change in vibrational, rotational or electronic energy of a molecule (see energy level). Chemists are concerned primarily with the vibrational Raman effect.

Brilluoin scattering – an interaction between an electromagnetic wave and one of the three crystalline lattice waves (phomoms, polarons and magnons).

Raman scattering – light scattered by interaction with vibrational and rotational transitions in the bonds between first-order neighbouring atoms.


The fluorescence lifetime refers to the average time the molecule stays in its excited state before emitting a photon. Fluorescence typically follows first-order kinetics.


Photo detectors

  • p-n junction photodiodes
    • non-linear function
    • slow, but low noise (no leakage current)
    • Large area, power efficient
    • small area, limited BW
  • Avalanche photodiodes (APDs)
    • One photon 10-100 e-h pairs
    • Multiplication: (M= 10 ~ 500)
  • Metal-Semiconductor-Metal detector
  • charge-coupled device (CCD)
    • a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value.
    • This is achieved by “shifting” the signals between stages within the device one at a time. CCDs move charge between capacitive bins in the device, with the shift allowing for the transfer of charge between bins.
  • Photo-resistor
  • Photo multiplying tubes
    • based on vacuum tubes. They can exhibit the combination of an extremely high sensitivity (even for photon counting) with a high speed. However, they are expensive, bulky, and need a high operating voltage.
  • Photoresistor

Lecture 4, waveguide based biosensor

Reflective Index, n = c/v

Snell’s law : \( \sin{\theta_{1}} n_1 = \sin{\theta_{2}} n_2 \)

Optical Fibre Classification

  • Extrinsic Sensors: to guide light/collect light
    • Absorbance
    • Reflectance
    • Fluorescence
  • Intrinsic Sensors: light property changes take place in OF
    • Evanescent wave
      • remove cladding of the OF
      • change in absorbtion by lambert law
      • \(I = I_0 e^{- \alpha c l }\)
    • Grated Fibres
      • Long period Grating (LPG) (10~100 um) by UV light
      • Measure change in peak frequency
      • No structure modification
    • tapered
      • Heated and streched OF, creating smaller waveguide
      • make OF more fragile
      • higher wave number, higher TM mode, evanescent field
      • Higher mode , higher sensitivity
      • Measure change in peak frequency

Lecture 5, SPR Biosensors:

Excitation of electron waves on metal surface by light


  • Grating Configuration
    • photons may interact with the sample
    • Not much in use
  • Otto configuration
  • Kretschmann Configuration
    • use prism
    • no airgap
    • most practical use


SPR sensitivity

\( S_{RI} = \frac{\delta Y}{\delta n_{ef}} \frac{\delta n_{ef}}{\delta n_{d}} \)

\( \frac{\delta Y}{\delta n_{ef}} = \text{machine performance}\)

\( \frac{\delta n_{ef} }{\delta n_{d}} = \text{test efficiency (e.g. binding)} \)

Minimum Resolvable Surface Coverage

The minimum resolvable change of molecular mass captured by the biorecognition elements

Minimum Resolvable Surface Coverage


Lecture 6: Electrochemical Sensing

charges effect on the electrode

E=E^0+RT/nF ln⁡(C_ox/C_red )

Electrochemical Cell

Electrochemical cell
Electrochemical cell

The cell consists of a working (W) and a counter (C) electrodes immersed in electrolyte, connected to a power supply. The reaction occurs between these electrodes. The third (reference) electrode (R) is an electrode with a stable electrochemical potential used in measuring the potential of the working electrode. This electrode also has contact with electrolyte but is surrounded by an insulating tube (Luggin tip or Luggin capillary) to prevent its polarization.


measuring change in potential when there is reaction


  • Amperomtry
  • voltammetry
  • Fix potential and measure the current
  • change in optential and change in current
    • Linear sweep voltammetery (LSV)
    • Cyclic voltammetry (CV)
      • Forward scan produces a current peak for oxidation of analyte; reverse scan
      •  reduction of product formed during forward scan current of reverse polarity;
      • Potential difference 59 mV for a reversible reaction; larger difference – non reversible reaction.
      • Used for solid electrodes.
      • CV
    • Anodic stripping voltamemetry (ASV)


Type of electrodes

Clark electrode

  • designed to measure blood PO2
  • The Pt electrode is a cathode (negative) and the Ag/AgCl is the anode (positive).
  • Oxygen is reduced at the cathode
  • O_2+ 4H+ + 4e-  →2H_2O

Glucose Example:

  • GOD-glucose oxidase
  • Glucose +O2  -> Gluconic Acid + H2O2
  • H2O2 > O2 + 2H+ + 2e
  • The Pt electrode is made positive (+650mv) with respect to the Ag/AgCl electrode (The electrons are on the other side of the equation with respect to  the Clark electrode

Nano material to improve sensitivity

Lecture 7: FET and Interferometric biosensors


Ion Selectie Electrode (ISE)

Glass electrodes for cations (normal pH electrodes) in which sensing element is a very thin hydrates glass membrane that generates transverse electrical potential due to the concentration-dependent competition between cations for specific binding sites selectivity is determined by the composition of glass.

Glass pH electrodes coated with a gas permeable membrane selective for CO2, NH3 or H2S. Gas diffusion causes the pH change of a sensing solution between membrane and the electrode, which is then measured.

limited number of commonly occurring ionic species


Metal Oxide Semiconductor (MOS)

  • apply voltage to the gate
  • minus carrier move the surface.
  • create channel for the current start to flow from Source to Drain.
  • channel regulated by the strength of the field


  • change the gate with electrolyte
  • change in electrical property will change the current in the channel

Ion-selective Field Effect Transistor (ISFET)

In the case of the ISFET, the gate metal electrode of the MOSFET is replaced by an electrolyte solution which is contacted by reference electrode

The metal part of reference electrode can be considered as the gate of the MOSFET. In ISFET, electric current (Id) flows from the source to the drain via the channel. Like in MOSFET the channel resistance depends on the electric field perpendicular to the direction of the current.

Characteristics Ion-specific electrodes (ISE) Ion-sensitive field effect transistors  (ISFET)
Measuring device input impedance High > 1012 Ω Low  104 Ω
Signal treatment Outside Integrated
Size Around 15cm, diameter 1.5 cm Quite small: 1.5×2.5 mm
Design Often with an internal liquid All solid
Cost High (low series) Low (microelectronic)

Enzymatic FET (EnFET)

differenttial measurement

  • limitation:
    • ionic strength
    • limited dynamic range

Example: silk stabilised glucose biosensor



Reflection/high RI : 180 phase change

  • Mach–Zehnder Interferometer (MZI)
    • 2 coupler
    • beam splitting
    • recombine
  • Michelson Interferometer
    • 1 coupler
    • moving mirror
    • different length
  • Fabry–Pérot Interferometer
    • reflection inside the cavity
    • require 2 reflection surface


Lecture 8 : Mass Sensitive Biosensors

Crystal Ocsillato/ Piezoelectric

  • base on surface acoustove wave ,
  • Apply voltage -> mechanical change
  • reversible process
  • AC voltage will produce

Longitudinal wave: particle move parallel to the direction of propagation.

Shear wave: particle move perpendicular to the direction of propagation.

Surface waves: Surface waves, also known as Rayleigh waves, represent an oscillating motion that travels along the surface of a test piece to a depth of one wavelength.

QCM: Quartz Crystal Microbalance

  • has resonance amplitude
  • Loaded QCM crystal exhibit shift in the resonance frequency

Sauerbery equation: frequency shift with mass change

\(\Delta F=-C_q F^2 \DeltaM_f \)

\(C_q=\frac{2}{\rho v} \)

Constants n, r and F are the material properties of the crystal. We can defined the crystal constant Cq .

  • higher fundamental frequency, higher sensitivity
  • limited by the thickness of the crystal
  • upto 100 Mhz’

Surface Acoustic wave devices (SAWs)

  • the substrate determine the wavelength
  • 100Mhz – 1 GHz (higher than QCM -> better sensitivity)
  • also sensitive t the environmental/electrical/temp change.
  • can be wirelessly coupled with antenna
  • change mass, change propagation velocity, change transit time

Atomic force microscope (AFM)

surface characterisation technique


  • vibrate at very high frequency
  • mass loading on cantilever will change the vibrating frequency
  • limited by thermal noise vibration
  • high damping factor in liquid
  • solution: tube micro-cantilever
  • not robust as SAW
  • 100kHz

Lecture 9 : Calorimetric Sensors



  • Thermocouples
  • platinum Sensors
  • Thermistor