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Biosensor, Surface Plasmon Resonance occurs when total internal reflection…

- - - - Signals are generated from the labeled antigens, higher conc, of the labeled antigens and lower the antigens, in the sample, bound to the primary antibodies, larger the signal produced
      - Antigens in the sample is inversely proportional to the fluorescent signal generated
  - - - Little signals are generated from the binding of analyte and ELISA (Enzyme linked immunosorbent assay) can be used to generate more signal -> higher sensitivity
        
        Quantification of hGH using optical fibers
        
        Optical fiber transmit light via total internal reflection (within the core of the fiber), and excited photons are transmitted through optical fibers
        
        Optical signals from immunoassays can be transmitted through the fibers and the intensity of optical signals are used to quantify the presence of hGH
        
        BRE can be immobilized onto the surface of the optical fibers core and light will pass though and interacts with fluorophores generating optical signals which will be transmitted back to the detector and separation of the excitation and emission photons may be required for clear identification, avoids inaccuracy
    - - Analyte and secondary antibodies are bounded to the surface/ other binding sites instead of BRE and blocking agents (BSA, casein) can be used to block off any available binding sites, by saturating the available surface binding sites.
    - - To prevent antibodies from binding to other structures besides the analyte, monoclonal antibodies can be used (reduced chances of cross reactions), increase incubation time (allow reaction to reach equilibrium -> stronger affinity between analyte and antibody) or affinity purification for polyclonal antibodies (removes antibodies which identifies interfering epitopes -> reduce adverse effects) can be done
        
        If the epitopes shared by 2 antigens are similar, cross reactions occurs and signal generated will not correspond to the concentration of analyte in the sample, either overestimation or underestimation, depending on the epitopes present in the interfering species.
    - - Excitation peak maybe too close to the emission peak in the spectrum thus fluorophore should be carefully chosen, one with larger stokes shift
  - - - Sandwich assay with a labeled secondary antibody is used to generate an optical signal, in the presence of hGH.
        
        Labels: Chromophore (colour signal), Fluorophore (fluorescence signal), enzyme (colour/ fluorescence signal) - attached to a secondary antibody via chemical bonding
        
        Steric hindrance due to label size -> when antigen-antibody complexes are closely packed, the use of bulky labels hinders the binding -> lower BRE event -> inaccuracy
        
        Features of an ideal label
        
        High activity -> low response time
        
        Stable under immunological reaction conditions (neutral pH level)
        
        low molecular weight -> minimize steric hindrance
        
        Having minimal impact on the biological activity of the antibody
        
        High intensity of emitted fluorescent light, large stokes shift and high photostability
        
        Chromophore: a molecule that absorbs certain range of wavelength in the visible light spectrum and the complementary wavelength is reflected and hence the colour. Higher concentration of the antigens, larger colour change is observed, strength of the colour change is measured by a spectrometer
        
        Amplification with enzymes (production of more products per enzyme) that catalyzes specific reactions requires suitable substrate, colourless substrate (for light to pass through and be reflected), to produce a colour or fluorescent product.
        
        Enzyme linked immunosorbent assay (ELISA)
        
        More sensitive than assays using fluorophore/chromophore; enzymes can catalyze multiple (colourless) substrates to intensify the optical signal (light intensity)
        
        Fluorophore: absorb photons of certain wavelengths and emits photons of longer wavelengths.
        
        Stokes shift: difference between between the wavelength of the peak absorption intensity and peak emission intensity. Larger stoke shift more desirable, in general, to differentiate between the excitation peak and emission peak
        
        Brightness: fluorophore’s molar extinction coefficient and quantum yield
        
        Photostability: Most fluorophores photobleached after some time under continuous illumination. This means that they lose the ability to fluoresce.
        
        Background signals: reduce background signals to reduce interference/ to avoid operating at the frequency range of the background signal
- - - - Resonant frequency of QCM, resonant frequency, f0, (Hz) = Vb/2d, where Vb is the bulk wave propagation velocity (m/s) and d is the plate thickness (m).
        
        Sauerbrey equation: when mass increases, plate thickness also increases as well as the change in resonant frequency, (final - intial)/(intitial) --> (resonant frequency decreases)
        
        Change in f = [final - initial] / [initial]
        
        usually final frequency is lower than the initial, due to the binding of analytes to the BRE, increasing the mass of the quartz crystal
    - - Based on the Sauerbrey's equation, the sensitivity of the biosensor strongly depends on the resonant frequency of the crystal plate. Thinner crystal plates (less stiff) are more desirable to enlarge the resonant frequency, while taking into account the mechanical stability. With larger resonant frequency, small changes in mass deposition, on the crystal plate, and larger change in resonant frequency can be detected.
  - - - Analytes bound to the sensor surface induces surface stress -> mechanical bending -> monitored by the optical detection method
        
        The Stoney's eqn: the dependence of radius of the curvature of the cantilever with the surface stress different between the responsiveness and passive sides o the microcantilever.
        
        The angle of bending affected by the surface stress, due to the bending of the analytes to the BRE. Higher degree of bending → smaller radius of curvature
        
        r - radius of curvature; v - poisson ratio; E - young's modulus; h is the thickness of the cantilever and Δσ is the difference in surface stress between the responsive and passive surface of the cantilever
    - - Similar to QCM, each microcantilever has its own resonant frequency (excited by an external piezoelectric actuation) which depends on the structure and material used
      - Mass deposition, frequency of the microcantilever experience a shift (Higher mass, resonance frequency decreases) and detection of this shift produced a signal
      - Binding of the analyte increases the mass on the cantilever surface, resonant frequency of microcantilever thus reduces
        
        m is the mass change of the microcantilever; k is the spring constant of the beam material; n is the factor associated with the geometry of the microcantilever; f0is the resonant frequency of the unloaded microcantilever; fm is the resonant frequency of the loaded microcantilever
    - - Binding of any mass results in signals generated in these mass sensitive devices -> undesirable signals generated
      - Blocking agents and reference-based detection can be used to reduced signals due to non-specific binding.
      - Amplification of signals: analytes with small mass cannot produced a sizeable signal in mass sensitive devices. Hence, sandwich assays with gold nanoparticles can be used to increase the change in mass, associated with analyte binding.
- - - - Selectivity: Ability of a biosensor to respond to a specific analyte or a group of analytes
  - - - Sensitivity: Measure of the responsiveness of the biosensor to the changes of analyte concentration (gradient of the response over concentration curve)
      - Linear range: Intensity of signal is directly proportional to the concentration of the analyte
      - Limit of detection: required lowest concentration of analyte in the sample solution for the biosensor to provide an appreciable signal
        (Usually mean blank response or 3 times the standard deviation of the noise in a blank)
        
        yLOD=yb+ksb
        LOD = yLOD/y
        y = ac
        LOD = yLOD/ac
        
        -> signal intensity at the limit of detection
        
        Signal intensity of blank measurement
        
        an arbitrary numerical factor chosen according to
        the confidence level desired. (Normally, k =3)
        
        standard deviation of the blank measurement
        
        depends on the background noise -> reduced noise, lower standard deviation
      - Reproducibility: Different independently manufactured biosensors produces a slight variation in response, when the same analyte is measured. However, the variation should not be too large such that the usefulness of the biosensor decreases, in terms of the measurement/detection. (indicated by the relative standard deviation)
      - Time factors: 1. Response time - how long the biosensor takes for a reading to be taken 2. Lifetime - the biological nature of the BRE limits the duration a usable biosensor
      - Other factors
        
        Multiplexing ability
        
        Able to distinguish and measure multiple analytes at one time?
        
        Sensitivity to pH and temperature
        
        Will the performance change drastically when pH or temperature changes?
        
        Portability
        
        Is
        the biosensor portable?
        
        In vivo application
        
        Is
        the biosensor implantable?
        
        Ease of Use
        
        Is the biosensor easy to use?
- - - - Every spot on the microarray represents different gene sequence from a DNA of known genome. Messenger RNA (mRNA) are transformed into cDNA by reverse transcription.
        
        cDNA from the first strain is cultured with a green dye while cDNA from the second culture is labelled with a red dye.
        
        These two are then combined and introduced to the microarray.
        
        When cDNA contains the sequence that is complementary to the probe, hybridization occurs, via complementary base pairing.
        
        Unbound cDNA are washed off
        
        After the hybridization step, microarray is scanned by a laser that is coupled to a special microscope and camera.
        
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    - - Mechanical deposition
        
        Oligonucleotides or DNA molecules can be synthesized and then mechanically deposited onto the functionalized substrate to make a microarray
        
        The inkjet printing head consist of a reservoir and different ink channels (millions of single nucleotides are found in the ink solution).
        The 4 reservoirs each contains a different nucleotide (A, T, G or C bases with deoxyribose sugar and phosphate group).
        The glass slide is functionalized for immobilization of the nucleotides.
        Each channel has a piezoelectric ceramic (When voltage is applied, piezoelectric will be displaced and the ink will be transferred from the reservoir, and the ink will flow into the channel in the absence of voltage, to be deposited simultaneously onto the glass slide.) to transfer the ink from the reservoir to the channel and be deposited onto the glass slide (non-contact printing).
        Specific nucleotide is deposited orderly, into a single stranded chain, at specific locations, but the spot size may vary considerably leading to lower reproducibility.
        
        Spot sizes can vary considerably, leading to low reproducibility
      - Photo-deprotection
        
        Repeated process of making, deprotection, reaction with nucleotide with photolabile protective group will create an oligonucleotide of desired length
        
        Highly reproducible; Expensive to produce custom arrays
        
        When exposed to light, through a patterned mask, photolabile groups may detach from the substrate, unexposed areas are protected and photolabile groups remains intact
        
        Photolithographic mask is used to determine the probe position on the array at which photo-induced deprotection of a previously deposited functionalised nucleotide occurs -> to attach the subsequent nucleotide to the growing oligomer
      - Digital micro-mirror device
        
        Reflective imaging system forms an ultraviolet image of the virtual mask on the
        active surface of the glass substrate.
        
        Two states of digital micromirror device
        
        Programmed chemical coupling cycles follow light exposure, and these steps are repeated with different virtual masks to grow desired oligonucleotides in a selected pattern.
        
        Replaces the chrome masks with virtual masks generated on a computer which
        are relayed to a digital micromirror array.
    - - Irregular spot morphology and position
        
        Bent or broken printing pins; different volume of printing solutions among the wells of a source plate due to evaporation
        
        Replace bent or broken pins; optimized printing humidity (minimise vapourization)
      - Doughnut shaped spots
        
        Surface irregularities
        
        Reducing the strike force of the printing pins and particles (impurities) in the printing environment and slide handling
      - Comets
        
        Loosely bound DNA reattached to the adjacent reactive areas in the slide surface to form comets
        
        Reduce the concentration of the DNA used and wash the unbound DNA thoroughly after the immobilization step.
    - - Logarithmic expression ratio (reduce to smaller numerical values that is easily readable)
        
        shows the relative expression level of each sample
        
        𝐿𝑜𝑔𝑎𝑟𝑖𝑡ℎ𝑚𝑖𝑐 𝑒𝑥𝑝𝑟𝑒𝑠𝑠𝑖𝑜𝑛 𝑟𝑎𝑡𝑖𝑜 =
        
        Use Excel for calculation due to the log base 2 to avoid conversion.
      - Each spot of the microarray contains many copies of the same DNA sequence comes from a unique gene
      - Different amount of binding by the respective fluorescent dye labelled cDNA on each spot of the gene would provide an indication of the level of expression for each sample (unbound sites will not fluoresce)
      - Calculation of the ratio provides an indication of the relative expression levels for each sample.
        
        Tk > 1 - gene expression of test samples higher than that of the reference sample Tk = 1 - equal expression Tk < 1 - gene expression for test samples lower than that of the reference sample
    - - Up regulation - expressed between 1 to infinity
      - Down regulation - compressed and mapped between 1 to 0
      - Fold change eliminates the inconsistency (up regulation map between 1 to infinity and the down regulation is mapped between 1 to 0) but it is discontinuous between -1 and +1.
        
        fold change = 0 when sites did not fluoresce; fold change => equal expression; positive fold change (>1) => high gene expression of test sample; negative fold change => high gene expression of reference sample
      - Tumor lung cell labelled green (Cy3), while healthy lung cell labelled red (Cy5); expression data for the heathy lung cell and tumor lung cell is plotted on the log scale shown above.
        
        Graph interpretation
        
        At a fold change of -5, cy5 is at least 5 times as bright as cy3 (red lines indicates the fold change)
        
        Areas around 1-> equal expression; up-regulation start from 2, down regulation start from -2
    - - The evolution of array technologies has enabled more features to be
        incorporated onto a microarray
        
        Microarray can detect multiple analytes simultaneously and hence is said
        to be a multiplexed analysis method.
        
        Due to its small size, multiple samples can be run in parallel and hence is
        said to be a high throughput analysis method.
- - - - Glucose (analyte) in the blood sample is catalyzed by the glucose oxidase (bio-recognition element) and the reactive species generated from the catalyzed reaction undergoes amperometry process to generate a signal (current flow) to be displayed.
        
        Catalytic reaction: Glucose + water + oxygen -- (glucose oxidase) --> gluconic acid + hydrogen peroxide
        
        The reaction is specific to glucose - measures the concentration of glucose present in the blood sample. Glucose oxidase, the BRE only binds to glucose for detection
        
        Amperometry (transduction reaction) means electrolysis on a microscale level
        
        2nd generation glucose biosensor: Artificial electron mediators removes the dependence on blood oxygen and are designed to undergo electrochemical reactions at lower electrode potentials -> reduces interference from the electroactive species in blood. For example, ferrocene requires an electrode potential of +0.18V to undergo oxidation.
        
        Catalytic reaction by glucose oxidase produces gluconic acid and ferrocene which will be oxidized to produce a current proportionate to the glucose conc.
        
        Examples of electron-mediators apart from ferrocene, o-Phenylenediamine, Hydroquinone
        
        Good electron mediators includes high stability, non toxic, high rate constant, electrode potential required for redox reaction near zero, moderate solubility and low molecular weight
        
        Does not break down during the reaction (stability), suitable for in vivo applications (toxicity), fast reaction -> reduce competition from blood oxygen to compete with the mediator, due to oxidative species (rate constant), prevent interference from reducible/ oxidizable species (electrode potential - near 0), prevent leaching out to solution or precipitation (solubility) and fast diffusion (molecular weight)
        
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        3rd generation glucose biosensor: enzymes are directly coupled to the electrode for direction electron transfer between the redox active centre to the electrode -> reduces interference by the electroacitve species in the blood
        
        Uses nanomaterials such as gold nanoparticles and carbon nanotubes to transmit electrical signals. Electrode materials such as mesoporous and organic conducting salts could possibly be used too
        
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        1st generation glucose biosensor: A constant voltage is applied between 2 electrodes to drive the reduction or oxidation reaction of substances resulting in a current flow. The electroactive species, H2O2, is oxidized when a voltage of +0.6V is applied at the electrode (H2O2 --> 2H+ + 2e- + O2). The amount of current flow is proportional to the glucose concentration.
        
        Possible interference from electroactive species such as ascorbic acid - vitamin C. At sufficiently high electrode potential, electroactive species found the blood sample can also be reduced (or oxidized), creating unwanted signals, resulting in an overestimation of blood glucose concentration and inappropriate administration of insulin -> might cause hypoglycemic coma or even death
        
        High electrode potential is needed to oxidize H2O2 -> other oxidizable species in blood sample can undergo oxidation resulting in unwanted signals
        
        present in the body due to drugs
        
        Other methods to reduce interference
        
        Use of membrane (of specific pore size or charges)
        
        Electroactive species could be excluded by the size or charge and multiple layers of different membrane can be used to exclude different electroactive species. E.g. Nafion has anionic SO3- groups and allows cations to pass through it while anions are not permeable
        
        Use of preoxidising layer
        
        Diminish electroactive interferences by employing a preoxidizing layer to convert the electroactive species into electrochemically inert forms before reaching the electrode surfaces. E.g. the use of ascorbate oxidase to oxidize ascorbic acid/ strong oxidizing agent such as MnO2 powders.
        
        [blue - immobilized enzyme, green- membrane or preoxidizing layer
    - - advantages: low cost, miniaturized and mass production
      - Screen printing technology involve the use of sequential deposition of different inks on the ceramic or plastic substrate with different screens and BRE can be immobilized subsequently
- - - - Physical barriers --> slower diffusion of analytes to BRE --> slower reaction time of biosensor
    - - Adsorption of other molecules occupy the sites for BRE binding -> interference or steric hindrance
      - Use blocking agents to block the available binding sites
  - - - Crosslinking
        
        Bifunctional agents such as glutaraldehyde and glyoxal can be used to form crosslinks (covalent bonds) between BRE. Formation of bonds between BRE and also between BRE and solid substrate.
        
        Simple method but high activity loss of BRE due to covalent bonding
        
        Risk of steric hindrance due to the orientation of the BRE -> actives sites becomes covered -> hinder binding of the analytes
      - Affinity
        
        Exploits strong and specific bonds between functional groups and an affinity tag. E.g. Biotin (vitamin H) and avidin (an egg protein). Enzymes and DNAs can be biotinylated at specific sites while avidin can be immobilized on the substrate.
        
        Allow control over the BRE orientation and prevents deactivation of BRE and steric hindrance - blocking of active sites
        
        Strong affinity bonding between the functional group and affinity tag gave control and oriented immobilization, but required specific groups such as biotin on the BRE
      - Covalent immobilization
        
        Functional/reactive groups (-NH2 , -OH, -COOH, -SH or even peptide bonds) on the surface of the BRE and solid substrate reacts with each other to form a stable covalent bond.
        
        If the substrate does not have suitable reactive groups, the surface can be activated with multifunctional reagents (carbodiimide and glutaraldehyde), to form a stable covalent bond (peptide bonds).
        
        Self assembled monolayer (SAM) - ordered molecular aggregates that form a monolayer of materials on a surface, due to the strong affinity between thiol groups and gold. Alkyl thiols with easily modified terminal functional groups (modified with other reagents) can be self assembled on the gold substrates. Self assembly occurs when hydrophobic and hydrophilic interactions drive the formation of a single layer of molecules on the substrate to form a highly ordered, stable and reproducible immobilization technique.
        
        There is a head (sulfur) (thiol) group on one end and a long hydrophobic tail on the other. Having a hydrophobic tail means that the tail part of the molecule doesn’t like water. When a solution of alkanethiol molecules is squirted on to gold, they self-assemble to form a thin layer. This is because the sulphur group is linked (attracted) to the gold and the hydrophobic tails pack together to push away the water in the solution.
        
        Terminal group X can be modified with other reagents
        
        Chemical binding - stable -> no desorption of the immobilized BRE, no diffusion barrier -> short response time, flexibility of immobilizing various BRE on the same type of the substrate, reproducible surfaces using SAM
        
        But there is a high possibility of denaturation -> alter the shape of active site -> high BRE activity loss
        
        Covalent bonds
    - - Adsorption
        
        Easy method - based on weak bonds; Van der Waal's forces, electrostatic forces, hydrophobic interactions, hydrogen bonding
        
        Apart from its simplicity, there is limited loss in BRE activity, but BRE could desorb due to the weak bonds formed and could lead to non-specific adsorption of other molecules/substances other than the analyte
      - Entrapment
        
        Physically trapping BRE in a matrix (membrane, gel, polymer or carbon paste)
        
        BRE trapped within matrix limits the loss in BRE activity and several BRE or the required mediators can be trapped in the same matrix
        
        Slow diffusion of analyte to BRE -> long response time and desorption -> possible leakage of BRE into the sample
        
        Matrix and capsule entrapment
- - - - T/Y mixer integrated with serpentine channels enhance fluid mixing by folding the fluid streams, depending on the fluid inertia forces -> works well at moderate reynolds number but at low reynolds no. where the laminar flow dominates -> inefficient
      - Chaotic mixer generate chaotic advection at microscopic scales by continuous stretching and folding, splitting and recombination of fluid -> They induce chaotic mixing even at extremely low reynolds no. thus significantly promote the mixing of fluid.
  - - - Re number is very low
        
        Movement of the fluid is predictable; in a single direction; parallel layers, wit no disruptions between layers, no lateral mixing
        
        Therefore, mixing becomes a challenge
        
        T/Y shaped mixer
        
        Chaotic advection
        
        To observe the efficiency of mixing -> fluid is dyed using different colours -> changes in colours indicates the mixing process -> Shorter distance required for mixing, higher the mixing efficiency.
    - - diffusivity of the of the molecules and the time required for diffusion can be determined by the Einstein equation
        
        H-filter (microseparator)
        
        Small particles (with higher diffusivity) will diffuse into the diluent stream (buffer solution), larger particles (with lower diffusivity) does not sufficient time to diffuse, at a given length, thus remain in the sample stream
        
        Buffer and sample solutions are brought into contact with a formation of a clear interface due to the low Re no. -> laminar flow; low Pe value -> molecules diffuse into the buffer solution, high Pe value -> molecules remain in the sample solution
        
        Given that 1 is the inlet for sample, 2 will be the inlet for the diluent, then 3 will be the waste outlet and 4 is the extracted particles' outlet
        
        Hydrodynamic filtration
        
        Fluid is pumped along the main channel and some liquids exits through the side channels; amount of fluid leaving the channel is carefully controlled.
        
        The constant removal of liquids into the side channels led to the alignment of particles against the (main) channel wall.
        
        Center of large particle further away from the channel wall -> flow straight -> follow the laminar flow; center of smaller particles are positioned along a stream line that enters the side channels
        
        Different flow speed in the side channels -> the center of mass of particles (different size) can be aligned to the stream of flow -> particles can be collected at different side channels
        
        Molecules transported by advection, not dependent on the diffusivity of the sample molecules
        
        Diffusion coefficient -> choose particles/ fluid with larger diffusion coefficient
        
        Residence time -> slow down the fluid flow rate such the residence time increase and more time is provided for diffusion. -> Increase the diffusion channel length, increase the residence time for diffusion to occur.
- - - - Total internal reflection occurs when angle of incidence is greater than the critical angle. The reflected light beam then leaks an electrical field intensity, evanescent wave, into the metal layer. Amplitude of evanescent wave decrease exponentially from the surface.
        
        Total internal reflection continues with increase angle of incidence (where angle of incidence > critical angle), generating evanescent waves repetitively for the excitation of the surface plasmon waves. (surface plasmon resonance)
        
        During excitation, energy is transferred to the surface plasmon waves, previously absorbed the evanescent wave, and surface plasmon resonance is observed as a drop in the intensity of the reflected light. Angle at which surface plasmon resonance occur is known as the resonance (SPR) angle.
        
        Surface plasmon resonance is very sensitive to the changes in refractive index, at the metal-sample interface.
        
        Binding of analyte or desorption of molecules from the metal surface lead to a change in the refractive index, resulting in a shift in the resonance angle, which will be detected and determines the presence of analytes in the sample.
        
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        The shift in the resonance angle is shown in the resonance curves and can be represented by the response unit (RU) in the response curve.
        
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        Non-specific binding: other molecules present in the sample can bind onto the available surface binding sites -> change in refractive index -> similar shift in resonance angle -> undesirable signals
        
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        Factors affecting refractive index: Temperature(usually a std) /density Wavelength
- - - - Immobilization of glucose oxidase - should only cover the working electrode for the potential to be measured reference electrode; Bubbles into the tubing could generate significant noise when reaches the electrode surface. Denaturation of the enzyme due to the enzyme drying up during the immobilization phase; enzyme leaching from the nafion membrane -> lower sensitivity
  - - - However, the electrolysis reaction lead to the electroactive species undergoing redox reaction, in the presence of high voltage applied. Vitamin C - ascorbic acid, can be reduced at high voltage, introducing a sizeable error in measuring any unknown solution (if the ascorbic acid is in the unknown solution).
- - - - C: number of unique optical barcodes that can be generated theoretically; N: number of intensities used; m: number of colours used in the scheme
    - - The presence of analytes is determined by the blue labelled secondary antibody or for the DNA barcodes; no complementary base pairing = no analyte
        
        Barcodes will be scanned using flow cytometry -> presence of the analytes would generate a specific fluorescence signal -> optical barcodes enable the identification of the specific analytes present
        
        Laser beam passes through the passage of analytes, in a thin channel, where the flow of the analytes in the sample is controlled by the compressed air (be detected and quantified). Filters will sort the analytes, depending on the type of fluorophores used). The laser beam that is directed at a hydrodynamically-focused stream of samples and will excite the fluorescently labelled analytes, emitting light of longer wavelength than the excitation state. Detectors then detect the wavelength of light, where signals are amplified, and data is analyzed based on the types of optical barcodes.
        
        Quantum dots vs organic dyes
        
        Emission Spectra
        
        quantum dots have narrow emission spectra -> will not overlap substantially -> multiple quantum dots of varying emission wavelengths could be used for the optical barcodes without overlapping peaks -> more distinguishable
        
        Absorption Spectra
        
        quantum dots have a wide absorption spectra -> different quantum dots can be excited using the same laser source -> multiple quantum dots (different colours) in a single microbead can be excited with one type of laser.
        
        Stokes Shift
        
        Wide stoke shift -> differentiate between the the absorption spectrum and emission spectrum
        
        Photostability
        
        High Photostability -> relatively same intensity after exposure to excitation photons for sometime -> longer lifetime
        
        Quantum dots are semiconductor nanoparticles; different sizes produce different optical properties; has superior optical properties to make optical barcodes
      - Biobarcode assay
        
        DNA consists of different base pairs, A, T, G, C, the sequence of base pairing can be used as codes to identify the presence of different analytes bound to the biorecognition element (multiplexed detection).
        
        DNA molecules can also be used for coding purpose
        
        A magnetic microbead is immobilized with a primary antibody to form a complex with the target molecules and a gold nanoparticle loaded with the DNA barcodes via a secondary antibody
        
        The complex is separated from the mixture, in the presence of an applied magnetic field and concentrated for release of the DNA barcodes from the gold nanoparticle and finally the detection of analyte on a microarray.
- - - - Example of an reaction of analyte (glucose) with BRE (glucose oxidase) as a catalyst
        
        The presence of products - H2O2 indicates the presence of glucose and the amount of gluconic acid and H2O2 is a measure of the concentration of glucose.
        
        Enzyme can be denatured due to the loss of the native conformation thus the active site in the enzyme, depending on the conditions, affecting the rate of reaction (reduced)
        
        Extreme pH/ high temperature -> denaturation of the BRE -> lost of natve conformation -> active site of the enzyme changes in shape -> cannot bind to the analytes
        
        Low temperature -> enzymes becomes inactivated. Therefore, an optimum condition is important for high enzymic activity -> increasing rate of reaction for efficiency
        
        Factors includes, extreme temperature and pH, ionic strength - a measure of the concentration of ions in the solution, the native conformation of enzyme depends on the ionic strength, which varies from enzyme to enzyme, and enzyme inhibitors - binding of inhibitors could prevent the analyte from binding with the enzyme's active site and hinders the enzyme from catalyzing the reaction.
        
        Substrate recycling amplification: Unlike PCR, substrate recycling achieves signal amplification by recycling analytes or byproducts of the biorecognition event.
        
        Without substrate recycling, low concentrations of analytes can
        only generate a weak signal.
        
        With substrate recycling, since analytes are regenerated , even
        low concentrations of analytes can generate a large signal.
        
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        Another enzyme lactate dehydrogenase can be used to recycle pyruvate into L-lactate.
        
        As the enzyme lactate dehydrogenase is able to recycle lactate, regenerated analyte can undergo an additional reaction to be catalysed by the Lactate oxidase again to obtain H2O2, thus, in very tiny concentration of L-lactate present, H2O2 can still reach detectable limits.
        
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      - Antibodies and antigens: Affinity between antibody (paratope) and antigens (epitope) allows each of the antibodies to bind to specific antigens (lock and key structure)
        
        Polyclonal and monoclonal antibodies
        
        Monoclonal: recognizes a specific epitope on the antigen
        
        Monoclonal antibodies are derived from a single cell line
        
        Polyclonal: recognizes multiple epitopes on the antigen
        
        Polyclonal antibodies are purified from inoculated mammals' serum
        
        Dissociation constants: Strength of binding between antibody and antigen is commonly expressed by the dissociation constant
        
        [Ab:Ag ], [Ab] and [Ag] are concentrations of the
        respective species at equilibrium
        
        Molecular Imprinted Polymers
        
        Artificial antibodies can be synthesized using the Molecular Imprinted Polymers (MIP) -> straight forward synthesis without the use of animal experiments
        
        Stable in extreme pH level and temp.
        
        Can be stored for years without a loss in activity
        
        target a wide variety of analytes -> possibility of imprinting against
    - - DNA double helix consist of Adenine (A), Thymine (T), Guanine (G) and Cytosine (C), complementary base pairing.
        
        Oligonucleotides - short sequence of nucleic acids, less than 50 bases long (detection is based on the DNA hybridization; affinity between A to T and C to G)
        
        Polymerase Chain Reaction (PCR) - create copies of DNA/specific segments of DNA, to reach the limit of detection of nucleic acid analyte (with sufficient amt of DNA fragments), but the bulky machinery is difficult to couple with biosensor for portable applications
        
        Step 1: denaturation- polymer chains of DNA are heated to break the hydrogen bonds (~95 degree C) Step 2: Annealing - temperature is lowered such that the primers can be attached to the denatured polymer chains (~60 degree C) Step 3: Elongation - temperature is increased for (Heat Resistant) DNA polymerase to function at optimal temperature for duplication (~70 degree C) Step 4: repeat the process to amplify the concentration of DNA (exponential growth)
        
        Thermal cycler
        
        Aptamers
        
        Single stranded DNA/RNA oligonucleotides, have high affinity and specificity towards a wide range of target molecules
        
        Relies on hydrogen bonding, electrostatic and hydrophobic interactions rather than Watson-Crick (complementary) base pairing
        
        Aptamers of favorable dissociation constants are selected from a library of random oligonucleotide sequence via the SELEX (Systematic Evolution of Ligands by
        Exponential enrichment) process
        
        SELEX: step 1 - incubation of the nucleic acid pool and the
        target, (2) separation of the bound from unbound molecules and (3) amplification of the bound molecules, with PCR
        
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        Aptamers are single stranded oligonucleotides that can bind selectively to proteins or small molecule compounds with comparable dissociation constants as antibody-antigen complexes