339
biotopics Separations
Purification is carried out initially to enable characterization and accurate quantification. Quantification of a desired product in a crude extract is almost always affected by the impurities, whatever method is tried, so that at least one stage of purification is needed. Techniques combining this separation and quantification in one step are therefore potentially useful for on-line sampiing of fermenters during productdevelopment work. There are interesting possibilities for collaboration between biochemical engineers and protein scientists. Characto'ization To industry, product characterization implies a degree of thoroughness that is not generally necessary in the science base. This is ultimately imposed by regulatory requirements which the science base does not have to meet. It has been reported that 750 separate tests were applied to Protropin - the recombinant
human growth hormone manufactured by Genentech. One of the attractions of small-molecule thera" peutics is their robustness and crystallizability, which makes purification more straightforward. Controlling the nature of the product and then detecting the known, as well as the largely unknown, impurities in recombinant protein therapeutics and reducing them to an acceptable level represents a major component of the cost of such medicines. There is a feeling that the recent development of new methods of analysis which offer sensitive and widely applicable ways of confirming conformation and chemical composition (e.g. 2DN M R and mass spectrometry) should begin to reduce, rather than compound, the arsenal of tests which companies are expected to apply. Perhaps someone should show convincingly that there would be no loss in confidence in product identity and quality by doing so, before the costs of applying new ana-
lyrical technologies become selfdefeating. There is room for new ideas here. People and ideas There is little doubt that the biotechnology industry looks to a strong science base for stimulating its own ideas. Most ideas arise from observations made somewhere else and so, although original ideas may well emerge within industry, the work that gives birth to them, as often as not, comes from outside without any planning on the industries' part. A strong, balanced international science base, with all countries putting in their fair share of fundamental work, is therefore essential. That same science base, at present, is the major source of trained people for the biotechnology industry and so its value is very great indeed. M i c h a e l J. G e i s o w Biodigm, 81 Kneeton Road, East Btidgford, Nottingham NG 13 8PH, UK.
Detection of nucleic acid hybridization using surface plasmon resonance The detection of nucleic acid sequences has been used in the diagnosis of genetic diseases, for forensic and paternity identification of individuals, to identify microorganisms and their resistance to drugs, and for mapping genomes. A DNA sequence can be detected by its ability to pair (through hydrogen bonding between complementary base pairs) to a D N A probe of complementary sequence (i.e. to hybridize). Conventionally a DNA probe is labelled with either radioisotopes, enzymes, fluorophores or small antigens. Hybridization of the DNA probe to its target is then detected by autoradiography, by chemiluminescence or by fluorescence 1. DNAprobe labelling and performing the assay can take 24-48 hours to complete. New technologies are currently being developed in the form
of biosensors to enable simple and rapid assays. Biosensors are devices in which a signal, usually electrical or optical, is generated when one species of biological molecule binds
to another; examples of such pairings are DNA- DNA, antigen-antibody, enzyme-enzyme substrate, hormone-hormone receptor and lectin-glycoprotein. Biosensors offer the potential of measuring DNA hybridization without the use of
workshop
Resonant wave Silver-coated
Laser light source
Detection array
Figure 1 When light at a specific angle is reflected at the metal surface between two media of different refractive index, surface plasmon resonance (SPR) can occur and the intensity of the reflected light is reduced.
¢) 1991,ElsevierSciencePublishersLtd (UK) 0167- 9430/91,/$2.00
TIBTECHOC'|OBER1991NOL 9)
340
forum Time scan
Angle scan
quires a two-hour reaction time, and the use of labelled probes s.
B
o
Angle
Time
Rgure 2 At time A, target DNA is added. The immobilized DNA probe binds to the target DNA, and the refractive index of the medium just abovethe metal surface increases. Theresonanceangle is shifted from A to B as shown inthe angle scan, and can be monitored in real time as shown in the time scan.
labels, and within minutes of simply adding the D N A sample of interest.
What is surface plasmon resonance? One of the most promising technologies for the direct detection of the binding of biological molecules exploits the phenomenon of surface plasmon resonance (SPR). Although SPR. was first described by Wood in 1.9022, it has only recently been used in biosensor technology 3. If light is totally internally reflected within a fibre optic, prism or hemicylinder, the energy of the light at the point of reflection generates an evanescent field in the overlying medium. The evanescent field extends approximately the distance of one wavelength of the light into the above medium, decaying exponentially with increasing distance from the interface. If a thin layer (-50 nm) of metal (usually silver or gold) is present on the surface of the hemicylinder, then the incident light again produces an evanescent field, but it also stimulates electrons at the metal surface into oscillations generating a surface plasmon wave (Fig. 1). Surface plasmon resonance will occur only when the light is at a specific angle of incidence. This angle, known as the resonance angle, is highly dependent on the refractive index of the medium immediately above the metal surface within the evanescent field. When resonance is established, the energy of the light is converted to the surface plasmon and so there is a reduction in the intensity of the reflected tight 4. The SPR, and consequently the resonance angle, are so sensitive to changes in the refractive index that D N A hyllBTECHOCTOBER1991(VOL9)
bridizations can be detected at the metal surface.
An SPR-based biosensor A biosensor for detecting D N A has been constructed by attaching a D N A probe to the metal surface. A laser generates a fan beam of light (780 nm) which spans a 6 ° angle (Fig. 1), and is focused on a 100 lan 2 spot at the metal surface. The intensity of the reflected fight is scanned every 3 ms by an array of photodiodes. As the D N A probe binds more sample or target DNA, the refractive index at the interface increases, and so the angle of reflected light required for resonance increases (Fig. 2). The change in the angle can be plotted against time to determine the rate of hybridization in real time (Fig. 2). Currently, a biosensor of this type can (within 5 min) detect 320 fg of a 97 mer and 24 fg of a 7.2 kb target after hybridization to a 50 mer probe immobilized on a 100 lan 2 sensing spot s . The sensitivity was calculated by determining the amount of radiolabelled D N A target bound to the metal surface and the corresponding change in the reflectivity for different concentrations of the D N A (double- and single-stranded D N A targets have been detected). By comparison, Southern blots can be used to detect as little as 100 fg of DNA. Other technologies (e.g. electrochemical 6 and microgravimetric 7) have been used to detect D N A hybridization, though their application is currently limited by poor sensitivity. A light-addressable potentiometric sensor (a device which uses light to measure changes in surface potentials) that can detect 2 pg of D N A has been described. However, it re-
Potential role o f the biosensor Biosensors that detect the binding of one molecular species to another are usefid for detecting the presence or absence of a particular D N A sequence in a sample. Some investigations also require the identification of the size and/or the electro-phoretic mobility of the target molecule, and in these instances methods based on gel electrophoresis may be more appropriate. The particular advantages of SPR-based biosensors are (1) rapid reading and (2) real-time kinetic analysis. Detection sensitivity approaches that of conventional methods, and simple protocols can be used because probe labelling is unnecessary. The operation of such a biosensor is suitable for automation and can be developed to detect hybridizations of a sample to a number of D N A probes simultaneously.
References 1 Pollard-Knight, D. (1990) Technique 2, 113-132 2 Wood, K. W. (1902) Philos. MaR. 4, 396-410 3 Nylander, C., Liedberg, B. and Lind, T. (1982) Sens.Aauators 3, 79-88 4 Kaether, H. (1988) Surface Plasmons on Smooth and Rough Sur[aces and on Gratings (Springer Tracts in Modem Physics,
Vol. 111), Springer-Verlag $ Pollard-Knight, D., Hawkins, E., Yeung, D., Pashby, D. P., Simpson, M,, McDougall, A., Buckle, P. and Charles, S. A. (1990) Ann. Biol. Ciin. 48, 642-646 6 Krznaric, D. and Cosovic, B. (1986) Anal. Biochem. 156, 454--462 7 Fawcen, N. C., Evans,J. A., Chien, L. C., Drozda, K. A. and Flowers, N. (1988) Sens. Technol. 4, 5-6 8 Kung, V. T., Pan~li, P. R., Sheldon, E. L., King, K. S., Nagainis, P. A., Gomez, B., Jr, Ross, D. A., Briggs,J. and Zuk, K. F. (1990) Anal. Biochem. 187, 220-227
Terek Schwarz Debra Yeung Edward Hawkins Paul Heaney Amerlite Diagnostics Ltd, Pollards Wood Laboratories, Nightingales Lane, Chaifont St Giles, Buckinghamshire HP8 4SP, UK.
Alex McOougaU Amersham lntemational plc, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, UK.
biotopics Separations
Purification is carried out initially to enable characterization and accurate quantification. Quantification of a desired product in a crude extract is almost always affected by the impurities, whatever method is tried, so that at least one stage of purification is needed. Techniques combining this separation and quantification in one step are therefore potentially useful for on-line sampiing of fermenters during productdevelopment work. There are interesting possibilities for collaboration between biochemical engineers and protein scientists. Characto'ization To industry, product characterization implies a degree of thoroughness that is not generally necessary in the science base. This is ultimately imposed by regulatory requirements which the science base does not have to meet. It has been reported that 750 separate tests were applied to Protropin - the recombinant
human growth hormone manufactured by Genentech. One of the attractions of small-molecule thera" peutics is their robustness and crystallizability, which makes purification more straightforward. Controlling the nature of the product and then detecting the known, as well as the largely unknown, impurities in recombinant protein therapeutics and reducing them to an acceptable level represents a major component of the cost of such medicines. There is a feeling that the recent development of new methods of analysis which offer sensitive and widely applicable ways of confirming conformation and chemical composition (e.g. 2DN M R and mass spectrometry) should begin to reduce, rather than compound, the arsenal of tests which companies are expected to apply. Perhaps someone should show convincingly that there would be no loss in confidence in product identity and quality by doing so, before the costs of applying new ana-
lyrical technologies become selfdefeating. There is room for new ideas here. People and ideas There is little doubt that the biotechnology industry looks to a strong science base for stimulating its own ideas. Most ideas arise from observations made somewhere else and so, although original ideas may well emerge within industry, the work that gives birth to them, as often as not, comes from outside without any planning on the industries' part. A strong, balanced international science base, with all countries putting in their fair share of fundamental work, is therefore essential. That same science base, at present, is the major source of trained people for the biotechnology industry and so its value is very great indeed. M i c h a e l J. G e i s o w Biodigm, 81 Kneeton Road, East Btidgford, Nottingham NG 13 8PH, UK.
Detection of nucleic acid hybridization using surface plasmon resonance The detection of nucleic acid sequences has been used in the diagnosis of genetic diseases, for forensic and paternity identification of individuals, to identify microorganisms and their resistance to drugs, and for mapping genomes. A DNA sequence can be detected by its ability to pair (through hydrogen bonding between complementary base pairs) to a D N A probe of complementary sequence (i.e. to hybridize). Conventionally a DNA probe is labelled with either radioisotopes, enzymes, fluorophores or small antigens. Hybridization of the DNA probe to its target is then detected by autoradiography, by chemiluminescence or by fluorescence 1. DNAprobe labelling and performing the assay can take 24-48 hours to complete. New technologies are currently being developed in the form
of biosensors to enable simple and rapid assays. Biosensors are devices in which a signal, usually electrical or optical, is generated when one species of biological molecule binds
to another; examples of such pairings are DNA- DNA, antigen-antibody, enzyme-enzyme substrate, hormone-hormone receptor and lectin-glycoprotein. Biosensors offer the potential of measuring DNA hybridization without the use of
workshop
Resonant wave Silver-coated
Laser light source
Detection array
Figure 1 When light at a specific angle is reflected at the metal surface between two media of different refractive index, surface plasmon resonance (SPR) can occur and the intensity of the reflected light is reduced.
¢) 1991,ElsevierSciencePublishersLtd (UK) 0167- 9430/91,/$2.00
TIBTECHOC'|OBER1991NOL 9)
340
forum Time scan
Angle scan
quires a two-hour reaction time, and the use of labelled probes s.
B
o
Angle
Time
Rgure 2 At time A, target DNA is added. The immobilized DNA probe binds to the target DNA, and the refractive index of the medium just abovethe metal surface increases. Theresonanceangle is shifted from A to B as shown inthe angle scan, and can be monitored in real time as shown in the time scan.
labels, and within minutes of simply adding the D N A sample of interest.
What is surface plasmon resonance? One of the most promising technologies for the direct detection of the binding of biological molecules exploits the phenomenon of surface plasmon resonance (SPR). Although SPR. was first described by Wood in 1.9022, it has only recently been used in biosensor technology 3. If light is totally internally reflected within a fibre optic, prism or hemicylinder, the energy of the light at the point of reflection generates an evanescent field in the overlying medium. The evanescent field extends approximately the distance of one wavelength of the light into the above medium, decaying exponentially with increasing distance from the interface. If a thin layer (-50 nm) of metal (usually silver or gold) is present on the surface of the hemicylinder, then the incident light again produces an evanescent field, but it also stimulates electrons at the metal surface into oscillations generating a surface plasmon wave (Fig. 1). Surface plasmon resonance will occur only when the light is at a specific angle of incidence. This angle, known as the resonance angle, is highly dependent on the refractive index of the medium immediately above the metal surface within the evanescent field. When resonance is established, the energy of the light is converted to the surface plasmon and so there is a reduction in the intensity of the reflected tight 4. The SPR, and consequently the resonance angle, are so sensitive to changes in the refractive index that D N A hyllBTECHOCTOBER1991(VOL9)
bridizations can be detected at the metal surface.
An SPR-based biosensor A biosensor for detecting D N A has been constructed by attaching a D N A probe to the metal surface. A laser generates a fan beam of light (780 nm) which spans a 6 ° angle (Fig. 1), and is focused on a 100 lan 2 spot at the metal surface. The intensity of the reflected fight is scanned every 3 ms by an array of photodiodes. As the D N A probe binds more sample or target DNA, the refractive index at the interface increases, and so the angle of reflected light required for resonance increases (Fig. 2). The change in the angle can be plotted against time to determine the rate of hybridization in real time (Fig. 2). Currently, a biosensor of this type can (within 5 min) detect 320 fg of a 97 mer and 24 fg of a 7.2 kb target after hybridization to a 50 mer probe immobilized on a 100 lan 2 sensing spot s . The sensitivity was calculated by determining the amount of radiolabelled D N A target bound to the metal surface and the corresponding change in the reflectivity for different concentrations of the D N A (double- and single-stranded D N A targets have been detected). By comparison, Southern blots can be used to detect as little as 100 fg of DNA. Other technologies (e.g. electrochemical 6 and microgravimetric 7) have been used to detect D N A hybridization, though their application is currently limited by poor sensitivity. A light-addressable potentiometric sensor (a device which uses light to measure changes in surface potentials) that can detect 2 pg of D N A has been described. However, it re-
Potential role o f the biosensor Biosensors that detect the binding of one molecular species to another are usefid for detecting the presence or absence of a particular D N A sequence in a sample. Some investigations also require the identification of the size and/or the electro-phoretic mobility of the target molecule, and in these instances methods based on gel electrophoresis may be more appropriate. The particular advantages of SPR-based biosensors are (1) rapid reading and (2) real-time kinetic analysis. Detection sensitivity approaches that of conventional methods, and simple protocols can be used because probe labelling is unnecessary. The operation of such a biosensor is suitable for automation and can be developed to detect hybridizations of a sample to a number of D N A probes simultaneously.
References 1 Pollard-Knight, D. (1990) Technique 2, 113-132 2 Wood, K. W. (1902) Philos. MaR. 4, 396-410 3 Nylander, C., Liedberg, B. and Lind, T. (1982) Sens.Aauators 3, 79-88 4 Kaether, H. (1988) Surface Plasmons on Smooth and Rough Sur[aces and on Gratings (Springer Tracts in Modem Physics,
Vol. 111), Springer-Verlag $ Pollard-Knight, D., Hawkins, E., Yeung, D., Pashby, D. P., Simpson, M,, McDougall, A., Buckle, P. and Charles, S. A. (1990) Ann. Biol. Ciin. 48, 642-646 6 Krznaric, D. and Cosovic, B. (1986) Anal. Biochem. 156, 454--462 7 Fawcen, N. C., Evans,J. A., Chien, L. C., Drozda, K. A. and Flowers, N. (1988) Sens. Technol. 4, 5-6 8 Kung, V. T., Pan~li, P. R., Sheldon, E. L., King, K. S., Nagainis, P. A., Gomez, B., Jr, Ross, D. A., Briggs,J. and Zuk, K. F. (1990) Anal. Biochem. 187, 220-227
Terek Schwarz Debra Yeung Edward Hawkins Paul Heaney Amerlite Diagnostics Ltd, Pollards Wood Laboratories, Nightingales Lane, Chaifont St Giles, Buckinghamshire HP8 4SP, UK.
Alex McOougaU Amersham lntemational plc, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, UK.
