USER DOCUMENTATION : BIODETEC 1) Introduction Biodetec is an optical biosensor for the study of molecular recognition and self-assembly processes on surface. Detection of these processes is based on the local measurement of refractive index, an intrinsic optical property of all materials. Since the refractive indices of biological and organic molecules such as proteins, lipids, DNA and alkanes are different from those of water, accurate measurement of the refractive index of a solution of these molecules allows the determination of their concentration. Biodetec uses optical waveguide chips to measure the refractive index at the surface of the waveguide. In these waveguides, part of the optical mode - the evanescent field - projects out of the substrate and into the solution on the waveguide (see figure). Changes in the refractive index of the solution within this region change the effective refractive index of the waveguide and this can be measured using optical gratings.

Optical gratings are used to couple light into and out of the waveguide. Light is shone on the grating at a specific angle of incidence and at a well-defined wavelength. Then, if the period of the grating is correct for the effective refractive index of the waveguide, the light will couple in. In Biodetec we use a ‘chirped’ grating, in which the period of the grating varies along its length. So for each effective refractive index of the waveguide, there will be one position on the grating where the light will couple in – this is known as the light pointer. We record this position using a CCD camera – the light that has coupled into the grating is coupled out by a second grating and falls on the camera. The position is used to determine the effective refractive index, and thus the mass of organic or biological molecules bound to the waveguide.

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Light pointer

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The Biodetec waveguide chips each have two measurement channels, which can be used in a number of different ways. Commonly, one of the channels is used to compensate for instrumental or temperature changes that can affect the measurement. This can be done in two ways: the two different measurement channels can each be coated with a different biochemical layer. Then both surfaces are exposed to the same solutions. Only one of the measurement channels reacts with the molecules present in the solution, however, changes due to temperature and refractive index fluctuations of the solution will be the same for both channels and can be subtracted out of the measurement. Alternatively, both measurement channels can be coated with the same chemical layer and measurements can be carried out using the double chamber cell. Different solutions are added to the different chambers of the cell. One chamber is used for the measurement of interest, while the other acts as a reference and allows compensation for instrumental changes or chip drift.

Reference

Signal

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2) Getting started: You should have: A keyboard, flat-screen monitor and mouse. The Biodetec instrument:

Three sample cells:

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The sample cell baseplate.

A box of waveguide chips. Assorted electrical leads.

Setting up: Connect the mouse, keyboard and monitor to the rear face of the instrument using the electrical leads provided. The sockets on the instrument are labelled to avoid confusion. Then connect the instrument to the mains electricity supply. The instrument is ready to be used.

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3) Hardware: The instrument itself: The biodetec instrument can be divided into two parts: the front of the instrument contains the optics and mechanical elements, while the electronics is in the rear of the instrument. Optics and mechanics:

Light source: a semiconductor laser is held in a temperature-controlled environment to produce monochromatic light of a stable wavelength, collimating optics produces a parallel light beam. Mirror: the mirror directs the light beam onto the waveguide chip at a well-defined angle Angle adjustment: a mechanism based on a cam and a stepper motor allows the incident angle of the light on the waveguide to be changed. CCD camera: the CCD camera takes an image of the waveguide chip. Fitting to the position of incoupling on the grating gives the changes in refractive index. Shutter: the shutter blocks the laser beam inside or outside the instrument at all times. When the external shutter is positioned over the window, the internal shutter is opened and the laser beam is blocked outside the instrument by the external shutter. When the external shutter is turned to one side, to allow pipetting or mounting of the sample cell assembly, the internal shutter blocks the laser beam inside the instrument.

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Sample cell: the sample cell aligns the waveguide chip with respect to the instrument optics and allows the waveguide surface to be exposed to different sample solutions in either flow-through or pipetting formats. The electronics: The electronics includes: the laser controller, which ensures a stable laser wavelength; the motor controller, which is used to adjust the position of the mirror; a frame grabber, which acquires images of the waveguide chip from the camera; a PC on which the Biodetec software runs. Sample handling: Three different sample cells are available for use with Biodetec: A flow-through cell, a pipetting cell and a double chamber pipetting cell.

1) Double chamber cell 2) Flow-through cell 3) Pipetting cell All three sample cells must be used with special silicone gaskets which seal the cell to the waveguide chip. The flow-through cell has a volume of about 5 microliters and must be used with a suitable pump – peristaltic or syringe – to exchange sample solutions and rinse the waveguide surface. The pump is attached to the cell using the two tubing connectors which screw into the two diagonal faces of the cell (see below). The pipetting cell is used for reactions in which new samples are added by pipetting. Removal of the sample from the cell is done by a pump, which is connected to a tube at the base of the cell. The use of a pump to empty the cell avoids touching the surface of the chip with the pipette, which may introduce artifacts. When pipetting into the sample cell the laser must first be switched off and the shutter moved to the open position. After all pipetting operations have been carried out, the shutter is closed and the laser switched on again. A minimum volume of 200 microliters is normally necessary to cover the chip surface The double chamber cell is similar to the pipetting cell: samples are added using a pipette, and the cell is emptied using a pump. However, a separate chamber is

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available for each of the two measurement gratings on the chip. In this way, identical surfaces can be prepared and then exposed simultaneously to different sample solutions. The responses of the two measurement channels can then be compared. A minimum volume of 100 microliters per chamber is normally necessary to cover the chip surface. Chip mounting: Before a measurement can be carried out a waveguide chip must be mounted on the instrument. First mount the chip in the sample cell using the mounting screws (see diagram below). All of the three sample cells – flow-through, pipetting or double chambre – can be used with the base-plate provided.

Mounting screws and tubing connector Flow-through cell Waveguide chip Baseplate

Once the sample cell, chip and baseplate have been assembled, mount the whole unit on the instrument. First turn the external shutter to one side then place the sample assembly over the measurement window. Three magnets in the assembly hold it firmly on the instrument, pressing on three steel balls that accurately position the chip with respect to the laser beam. Attach the cell to the external pump using the tubing connectors. Finally, reposition the external shutter over the measurement window before starting measurements.

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1) Shutter turned to one side. 2) Assembly mounted on instrument. Shutter ready for measurements. Safety: Laser safety: Biodetec uses a class 3B infrared laser (785 nm). The light from the laser is almost invisible to the naked eye and can permanently damage your eyesight. For this reason Biodetec is equipped with a shutter, which always blocks the laser beam: either inside the instrument or outside the instrument. This shutter should never be removed or modified, nor should the instrument be opened or disassembled while the laser is on. Electrical safety: There are no parts that can be changed or repaired by the user inside the instrument housing. The instrument housing should not be opened while the instrument is plugged in to the mains supply.

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4) Waveguide chips: The waveguide chips used in Biodetec have a TiO2 waveguide on top of a polycarbonate substrate. Optical gratings in the polycarbonate chip are formed by injection moulding and the whole surface is then covered with approx 130nm of TiO2 by sputtering under vacuum.

Waveguide chip with the four chirped

gratings used in Biodetec measurements. Before use, the waveguide chips should be cleaned using one or both of the following methods:

a) sonication in a strong detergent (e.g. Extran) for 3 minutes and then rinsing many times with deionised water. Finally, sonication for 3 minutes in deionised water.

b) plasma cleaning: 3 min, 250 W, microwave plasma, 50% oxygen content, at 250 mTorr. After plasma cleaning the chips are kept in water until use, which must be on the same day as the plasma cleaning.

The waveguide surface can be functionalised in a number of different ways including silanisation and physisorption of proteins on the surface. For all functionalisation processes, all organic solvents should be avoided apart from alkanes and some alcohols for short periods of time. Strong acids and bases should also be avoided as they attack the waveguide layer.

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5) Measurements Getting started Setting up: First set up the instrument as described in section 2. Mount a new waveguide chip in a sample cell as described in section 3. Then mount the sample cell assembly on the instrument. Switch on the instrument using the button on the top right. You will have to wait for a minute or two while the computer boots up. Once Windows has started, you will be prompted for a network password. If you do not wish to log on to the network click cancel. Start the Biodetec software by clicking on the icon. Chip characterisation: The software opens with the screen in the figure below:

Opening screen in Biodetec software. On the right hand side of the screen there are three windows. These will display the data obtained by the instrument during calibration and measurement. On the left hand side of the screen there are three pages: Chip testing, Measurement and Noise. The software should open at the page ‘Chip testing’. If not, then move to this page.

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Calibrate the chip by clicking on the green button ‘Start characterisation’. First the instrument will then scan through a wide range of angles to locate the position of the peaks. Then the exposure time of the camera will be adjusted. Finally a calibration curve will be recorded, which relates the observed position of the peak to the effective refractive index of the waveguide. This calibration curve will be fitted to a 7th order polynomial curve. You can observe the characterisation process in the three windows on the right hand side of the screen. The top window is an image of the chip as seen by the CCD camera. The centre window shows the peaks and the fits to the peaks obtained. The third window shows the positions of the peaks during the calibration. Once the calibration curve has been recorded, it should be saved using ‘file’ and ‘save chip data file’.

After characterisation of the chip. If large deviations are observed between fit and calibration curve, the chip should not be used for measurements. If necessary, the chip characterisation can be stopped at any time using the button ‘abort’.

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Measurements: To start a measurement, first change to the ‘measurement’ page. Using the topmost slider, choose the working point of the measurement. The working point is the point on the calibration curve where the measurement will start. It should be in a region where calibration data and fit correspond well for both red and green curves. Using the next slider down, choose the time interval between data points. This time can take any value between 100ms and 12000ms in units of 100ms. When choosing the time interval it is worth bearing in mind that a shorter time interval will result in larger measurement files and more noise on the data. A longer time interval will result in less noise and smaller files but the time resolution will clearly not be as good. The time interval between data points can be changed during the measurement. Once the working point and time interval have been set, start the measurement by clicking on the button ‘start’.

During a measurement. When pipetting into the sample cell, it is important to first interrupt the measurement by clicking on ‘break’. Then open the shutter. Once pipetting operations are over, close the shutter again and click on ‘cont.’ to restart the measurement. While the measurement is interrupted, the time of the measurement will continue to be recorded by the software, but no data will be saved.

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To end a measurement click on ‘stop’. Once the measurement is finished, it should be saved using ‘file’, ‘save measurement’. Display options: Zoom During a measurement you may wish to zoom in on a certain portion of the measurement data. This can be done using the ‘plot/scale options’ on the measurement page. First change from ‘auto’ to ‘manual’. Then enter the y and x values that define the window you would like to observe. Click ‘set’ to apply these values. If you wish to see the whole measurement curve again, click on ‘auto’. Color It is possible to change the color of one of the measurement curves during the measurement using the ‘color’ button. This feature can be useful to mark different events (e.g. addition of a ligand or protein) during an experiment. Note that the change in color is not marked in the final ASCI file of the measurement. While the ‘color’ window is open, the software does not record data. Window At the bottom right of the screen there are three buttons labelled ‘camera’, ‘peaks’ and ‘data’. These buttons may be used to change the different windows displayed during the measurement. General points for good experiments: Drift: At the start of the measurement you may well observe a slight drift on the effective refractive index of the waveguide. In the ideal case this drift should be identical for both signal and reference curve, so that, when the two channels are compared, the result is a straight line. You may, however, see a small difference in the drifts of the two measurement channels. This drift will result in small spurious signal when the two channels are compared. The size of the drift that is acceptable will depend upon the magnitude of the signal that you expect to observe in your measurement, and the time over which this binding and displacement event takes place. If the drift of the waveguide is slightly too big, it is often worth waiting for a few minutes, since the drift often decreases rapidly at the start of the measurement. Changing solutions: Biodetec measures changes in the effective refractive index of the waveguide. These changes can either be caused by binding (or displacement) of molecules at the surface of the waveguide or by changes in the bulk refractive index of the sample solution. Even a very small change in the refractive index of the solution (e.g. caused by small differences in concentration or temperature) will result in a large signal, so it is important to be aware of these effects. It is often useful to use one solution as a reference, or baseline solution, and to return to this solution in between binding events at the waveguide surface.

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It is important not to let the chip surface dry out when changing solutions as this can lead to irreversible binding of proteins and salts to the waveguide surface. Advanced options: Average data points In normal use, the software measures one data point every 100ms. However, if the time interval between data is set to more than 100ms, all measured data points in this time interval will be measured and the mean value of these measurements will be given as the recorded data point. This results in less noise for longer time intervals between data points. It is possible to remove this feature by clicking in the box ‘average data points’. In this case the noise will be independent of the time interval between data points. Noise It is possible to directly observe the noise on each measurement channel on the page ‘noise’. The slider ‘number of RMS points’ can be used to change the number of points used to calculate the noise.

The noise obtained during the above measurement. Video options The live video window can be stopped or started using the button on the bottom frame. When the live video is off, individual images can be grabbed using the button ‘grab single’.

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7) Data: Data files from the Biodetec software are tracked using the programme “HTML database”. The data files are listed by date and for each file an image of the screen when the file was saved is kept. The images are saved using the file name without the extension, so it is important that the file names be different if the images are important. Data are stored as ASCII files and can be displayed and manipulated using any suitable graph/spreadsheet software e.g. Excel, Igor, Origin. Data files can also be displayed using the programme “Data view”. Once the file has been chosen, the two curves are displayed in the main window. The difference curve is displayed in the lower window. Sections of the curve can be selected or zoomed in on using the buttons in the top bar. The section of curve displayed can be moved using the hand button.

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