Molecular Forensics. Edited by Ralph Rapley and David Whitehouse Copyright 2007 by John Wiley & Sons, Ltd.

respectively), and voice and signature recognition (6% and 2%, respectively). Biologically-based technologies in forensic sciences are mostly limited to polymerase chain reaction (PCR)-based DNA 'fingerprinting'. Whilst DNA can be matched against databases relatively easily, in the absence of a match, tissue samples, body fluids or stains may still reveal vital intelligence information useful for fitting the person's biometric or behavioural profiles or for gathering additional forensic information. DNA assays are most suitable for matching and identification of individuals, whilst protein and metabolite profiles are more representative of the state of health, lifestyle, behavioural patterns, sample origin (tissue/organ/time), the severity and type of trauma or the cause of death. DNA profiling is incapable of monitoring these. mRNA profiling may be, but mRNAs are restricted to their respective tissues and cells (and are very unstable molecules), whilst proteins and metabolites are often secreted and can be detected/measured in body fluids, e.g. blood/serum/urine/saliva. Single protein assays have been proposed for forensic analysis before, but so far the research has been fragmented and until very recently no technical capabilities existed for highly parallel and quantitative analysis of proteins and scene-of-crime assays.

Affinity immunoassays have been widely used for achieving specific and sensitive detection of analytes of interest. A wide range of existing assay types includes colorimetric (Khosravi et al., 1995; Garden and Strachan, 2001; Moorthy et al., 2004), radiometric (Ahlstedt et al., 1976; Yalow, 1980; Raja et al., 1988), fluorescence (Kronick, 1986; Dickson et al., 1995) and chemilu-minescence (Rongen et al., 1994) detection methods, each incorporating their own form of labels, such as radioisotopes for radiometric tests or organic dyes for fluorescence-based assays. The performance of these tests is usually restricted to central laboratories because of the need for long assay times, complex and expensive equipment and highly trained individuals, but there is an ever-increasing market for new, faster, more accurate and cost-effective diagnostics that can be supplied in kit format for use in the field. The ability to multiplex such assays is also highly desirable, allowing for the simultaneous detection of more than one analyte in a given assay.

Liquid chromatography assays

Size exclusion chromatography permits the separation of molecules by physical size and thus can be harnessed for use in a potentially very attractive immu-noassay format. The assay can be in the form of a large-scale gel filtration set-up utilizing a column packed with gel media of chosen fractionation range (e.g. Sephadex®), or a small - scale set-up by way of MicroSpin® (both from GE Healthcare) columns. Gel filtration media separate molecules according to size, with larger molecules being eluted first, followed by smaller molecules in order of their size. This can be harnessed in an immunoassay format by exploiting the size differences between antigen-antibody complexes and unbound antibody and antigen. Antibody molecules are -150 kDa; other proteins are of variable size but far larger than peptides, which are -1-3 kDa, and small-molecule drugs (<1 kDa). Therefore a column containing gel with a fractionation range capable of resolving molecules of -150 kDa (antibody) from molecules of larger size (antigen-antibody complexes) would be suitable for use in such an assay format. The assay is compatible with both competitive and non-competitive formats, both of which can be designed a number of different ways:

1. Competitive:

a. Fluorescently labelled reference competes for antibodies with sample antigen. Fluorescence intensity is inversely proportional to sample antigen concentration.

b. Fluorescently labelled sample competes for antibodies with unlabelled reference. Fluorescence intensity is proportional to sample antigen concentration.

2. Non-competitive:

a. Fluorescently labelled antibodies are mixed with sample to form labelled antibody-antigen complexes. Fluorescence intensity is proportional to sample antigen concentration.

b. Fluorescently labelled sample is mixed with antibodies to form labelled antibody-antigen complexes. Fluorescence intensity is proportional to sample antigen concentration.

Given the small size of peptides and small drug molecules, labelling antibodies may pose problems in that the column may not be capable of resolving the relatively small differences in size between unbound antibody and antibody-antigen complexes. In this instance labelled antibody would provide a potentially misleading result as to the amount of sample antigen concentration and an overall low level of detection. A better choice would be to label antigen (either sample or reference), which will be dramatically different in size to antibody within the system, and thus if the column was to elute some free antibody with antibody-antigen complexes then this would not provide such a misleading result.

Eluted buffer should be collected from the column up until the point where only antibody-antigen complexes have had a chance to elute. This collection can then be scanned with a spectrofluorimeter. Standard curves should be made for a range of known sample antigen concentrations by using single antigen-antibody pairs. Following successful completion of this, a multiplex assay may be attempted, which can be achieved though using a range of fluorescent tags for different analytes. For multiplexing, competitive assays with fluorescently labelled reference are the only suitable option because they provide the simplest way to assign different fluorescent tags to specific analytes. Miniaturizing the assay results in a format more applicable to the field-based studies. A microspin format (e.g. similar to Bio-Rad Micro Bio-Spin columns) or small cartridge-like gravity-flow format (e.g. similar to NAP columns from GE Healthcare) with columns containing the same gel capable of quantitative separation of antigen-antibody complexes from unbound antibody presents such a solution. A portable spectrofluorimeter could then be used to check for fluorescence intensity as described above, making the format applicable for scene-of-crime applications.

Immunochromatography assay format

Otherwise known as lateral flow or strip tests, immunochromatographic assays exhibit a number of highly desirable benefits, including a user-friendly format, rapid sample turnaround, as well as being relatively inexpensive to produce. Such features make them ideal for affordable field-based or point-of-care testing. Pregnancy tests are an example of an immunochromatographic assay designed for qualitative determination of human chorionic gonadotrophin (hCG) in urine for early detection of pregnancy. Lateral flow devices have been applied successfully to a wide range of detection applications, including aflatoxin B1 in pig feed (Delmulle et al., 2005), botulism neurotoxins in foods (Sharma et al., 2005) and drugs of abuse (Niedbala et al., 2001). In their simplest form, lateral flow tests consist of a porous membrane strip such as nitrocellulose that has a band of capture antibodies immobilized at a discrete point across its width (Qian and Bau, 2004). This mixture diffuses through the membrane towards the capture line where hybridization occurs, detectable by standard fluorescence detection methods. A control line is added to the membrane after the capture line, consisting of immobilized antibodies that bind to the reporter but not to the analyte of interest. Lateral flow assays are compatible with competitive and non-competitive immunoassay formats, outlined below:

1. Competitive:

a. Fluorescently labelled reference competes with sample antigen for antibodies discretely spotted on a membrane strip. Fluorescence intensity is inversely proportional to sample antigen concentration.

b. Fluorescently labelled sample competes with unlabelled reference for antibodies discretely spotted on a membrane strip. Fluorescence intensity is directly proportional to sample antigen concentration.

c. Fluorescently labelled antibody is added to sample and then run on membrane strip with reference antigen discretely spotted at a certain point. Fluorescence intensity is directly proportional to sample antigen concentration.

2. Non-competitive:

a. Fluorescently labelled sample antigen binds to antibodies at a discrete capture line on membrane strip. Fluorescence intensity is directly proportional to sample antigen concentration.

b. Fluorescently labelled antibody binds to sample antigen spotted at a discrete capture line on a membrane strip. Fluorescence intensity is proportional to sample antigen concentration.

In practice neither of the non-competitive forms of the assay are ideal because they require the sample to be either fluorescently labelled or spotted onto the membrane strip prior to testing. Minimum sample preparation should be the focus and for this reason the first and third competitive formats are the most appealing. Competitive forms of lateral flow tests can also be used for small-molecule analytes with single antigenic determinants that are incompatible with sandwich forms of the assay (Qian and Bau, 2004). Predictive tools have been developed by computer modelling, allowing simulation and optimization of a device and reducing the number of laboratory experiments needed in the development of lateral flow devices (Qian and Bau, 2003, 2004).

The membrane strip can be scanned, with intensity of fluorescence at the capture line quantitatively indicating the presence or absence of sample antigen. The assay may first be assessed using single antigen-antibody pairs for which standard curves can be plotted for a range of known sample concentrations, followed by a multiplexed approach once conditions for each analyte have been optimized. Multiplexing may be achieved by spatially separating the capture lines for each analyte or by using a single capture line and assigning a fluorescent tag of different colour to each analyte of interest.

Immunochromatographic assays are highly suited to incorporation into kit format that would usually consist of a mould essentially enabling the user to plug-and-play by simply adding a sample to a defined region of the membrane. Such kits offer standardization of use each time for position/sample application/detection, and can be achieved with minimal effort.

Other formats

Quartz crystal microbalance. The quartz crystal microbalance (QCM) is a simple and convenient method of quantitatively measuring very small masses in real time. It is a form of acoustic wave technology, so called because an acoustic wave is the mechanism of detection. The velocity or amplitude of the wave can be changed as it passes through the surface of the material, and such changes can be detected by measuring the frequency or phase characteristics of the sensor. Any changes can be correlated to physical interactions occurring on the surface of the sensor, such as binding of sample analyte to surface-immobilized antibody, which would result in a frequency decrease due to a mass increase from the biological interactions (Thompson et al., 1986; Muratsugu et al., 1993; Sakai et al., 1995). Such devices are classified by the mode of wave that propagates through or on the substrate, and, of the many wave modes available, shear-horizontal surface acoustic wave (SH-SAW) sensors are best as biosensors due to their superior ability to operate with liquids (Drafts, 2001). A special class of these is the Love wave sensor, which consists of a series of coatings on the surface of the device, including a final coating with biorecognition capability. The Love wave sensor has demonstrated excellent sensitivity (Gizeli et al., 1992, 1993; Kovacs and Venema, 1992; Du et al., 1996) and the ability to detect anti-goat IgG in solution in the concentration range of 3 x 10-8 - 10-6 m (Gizeli et al., 1997). The QCM technology has been applied to a number of other fields, such as detection of a class A drug (Attili and Suleiman, 1996) and mutations in DNA (Su et al., 2004), and is commercially available from a number of providers, e.g. Attana Sensor Technologies Ltd [] or Akubio Ltd []. Figure 13.1g summarizes the basic principles behind the technology.

Rupture event scanning. The QCM technology is used in another form of biosensor known as rupture event scanning (REVS). However, rather than being used to measure mass increase, as is the case with other QCM-based detection systems such as the Love wave sensor, a piezoelectric substrate is used to detect the binding and estimate the affinity of analyte binding to antibodies covalently attached to the surface by detecting acoustic noise produced from the rupturing of bonds between antigens and antibodies. By applying an alternating voltage to gold electrodes on the upper and lower surfaces of a disc of crystalline quartz, and monotonously increasing the voltage and thus the amplitude of the transverse oscillation of the QCM, Cooper et al. (2001) demonstrated a novel way of directly, sensitively and quantitatively detecting virus particles bound to specific antibodies immobilized on the QCM surface. Figure 13.1h depicts the general principles involved in REVS. Both the Love wave sensor and REVS are suitable for forensic (field and laboratory) applications and have the additional advantage of providing label-free detection of molecules, allowing interactions to be monitored between unmodified reactants.

Surface plasmon resonance and BIAcore. Another label-free approach to assaying an analyte of interest in a sample is by way of the BIAcore system. The BIAcore system is based on surface plasmon resonance (SPR), an optical phe-


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