Replacing biological assay components with synthetic analogues

In conventional sandwich ELISA immunoassays the biological recognition of the analyte is based on antibodies, while the signal transduction and amplification of the antigen-antibody recognition events is provided by the enzyme label. However, for actual technological applications, the use of proteins (antibodies and enzymes), which are delicate, biological-origin molecules with narrow pH and temperature optimum, high chemical sensitivity and subsequently short shelf-life, not to mention the relatively high preparation cost, and restricted amount that can be generated is often limiting. Therefore, in the past decades increasing efforts were done to replace antibodies and/or enzyme labels with in vitro generated, synthetic analogues in immunoassays. These analogues provide the same basic functions as their biological counterparts, but they are more robust and cost-effective. They provide easily adjustable properties, often expanded with novel characteristics.

4.2.3.1 Synthetic receptors

Although antibodies offer a wide range of applications for almost half a century and thus have become indispensable in most diagnostic tests, they are often limited in terms of ruggedness and cost-effectiveness. General understanding of the molecular recognition processes in the past decades permitted to imitate biorecognition, resulting

31 in affinity reagents including RNA and DNA aptamers, peptides, and a variety of protein scaffolds^[171]. Unlike antibodies, they are synthesized via an in vitro process, hence their properties can easily be changed on demand, they can be modified chemically, and their accurate and reproducible synthesis ensures little or no batch-to-batch variation. As no cells are involved in this process, toxins or molecules that do not elicit a good immune response can be target molecules as well. These artificial recognition sites may be substituted for antibodies in bioaffinity assays.

In this thesis two synthetic receptor families were applied, aptamers and molecularly imprinted polymers

Aptamers

Aptamers are in vitro generated, short, single stranded oligonucleotides (both DNA and RNA) that selectively bind to target compounds ranging from small molecules to macromolecules. Their name is derived from the Latin word ‘aptus’ meaning ‘to fit’^[172].

The identification method of oligonucleotide sequences with unique binding properties to target molecules, a technique called SELEX, was described in 1990^{[172] [173]}. SELEX, systematic evolution of ligands by exponential enrichment, is a technique for screening very large, 10¹²‒10¹⁴ random sequence, combinatorial libraries of oligonucleotides by an iterative process of in vitro selection and amplification. This technique made the isolation of aptamers with the capacity to recognize virtually any class of target molecules with high affinity and specificity possible.

The dissociation constant of aptamer-ligand complexes is similar to that of antibody-antigen interactions, however, aptamers are superior to antibodies in many aspects.

Besides the obvious advantages of the in vitro selection, aptamers, owing to their nucleic acid composition, are amenable to well controlled chemical modifications, have good chemical stability and are easy to handle. Their main advantage over monoclonal antibodies is their resistance to thermal denaturation. They are robust and easy to synthesize. Aptamers have great potential in bioanalysis^[174][175], most of the routine immunanalytical methodologies can be easily adapted to detect aptamer-ligand interactions^[176] and thus aptamers can potentially fulfil molecular recognition needs in immunoassays.

A possible limitation of their use as diagnostic receptors can be, however, that oligonucleotides are susceptible for the ubiquitously present nuclease enzymes.

Therefore, lately various attempts were done to generate aptamers built from modified nucleotides with enhanced nuclease resistance^[177]. Spiegelmers^[178], based on the enantiomers of natural RNA and DNA molecules, i.e. L-ribose or L-2′-deoxyribose units, are an aptamer variety showing complete resistance to enzymatic degradation.

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Molecularly imprinted polymers

A promising technique to generate fully synthetic recognition sites is molecularly imprinted polymers (MIPs). Molecular imprinting is a method to generate materials with “molecular memory” by performing a polymerization of suitable functional monomers in the presence of the target molecule, acting as a template.

The concept was established in 1972 by Wulff and Sarhan when introducing template molecules during polymerization what they then called “host-guest polymerization”^[179]. The preassembly of the functional monomers around the template is conserved by the polymerization, hence, after the template is removed, recognition sites complementary with shapes, sizes and orientation to the template remain within the polymer matrix. This straightforward strategy enables chemists to generate synthetic polymers, molecularly imprinted polymers that are selective towards a specified template via complementary non-covalent binding sites, i.e. ionic, hydrophobic or hydrogen bond interactions^[180].

Molecularly imprinted polymers could easily compete with antibodies in terms of cost and complexity of synthesis, stability and mechanical properties as well as the range of target molecules. However, molecular imprinting still faces challenges when imprinting large, delicate biomacromolecules, such as proteins^[181], and often fails to work as synthetic antibody^[182]. So far the affinity and catalytic activity of MIPs have in general been lower than those of their biological counterparts^[183].

Although the bulk synthesis method produces excellent results in generating MIPs for recognition of low molecular weight compounds^{[184][185][186][187]}, the macromolecules become entrapped in the polymer material, which hinders both removal and rebinding of the target. Thus, an essential prerequisite of protein imprinting is to create binding sites accessible for the target from the solution phase, i.e. to have binding sites confined to the surface of the MIPs. To realize this requirement “surface imprinting” techniques have been emerged lately, based on polymeric micro- and nanostructures. A few examples include protein surface imprinting of nanometer thin films^{[188] [189]}, microrods^[190][191] or microbands^[192]. Surface imprinting is inherently advantageous to minimize non-specific interactions as well, because it reduces the contact area between macromolecular targets and bulk polymeric material.

4.2.3.2. Synthetic labels

Similarly to antibodies, the enzyme label can also be replaced by synthetic materials.

The attractive amplification and multiplexation properties of metal and semiconductor nanoparticles make them ideal labels for bioaffinity assays. The unique and attractive properties of nanomaterials have paved the way for the development of highly sensitive electrochemical diagnostic devices.

33 Nanoparticle-labelled affinity reagents

Nanoparticle labels provide excitingly new possibilities for advanced development of new analytical tools and instrumentation, especially on the field of electrochemical immunoassays. Antibodies or other affinity reagents labelled with nanoparticles (antibody ‒ nanoparticle or affinity reagent ‒ nanoparticle conjugates, respectively) can retain their bioactivity due to the similar dimensions of nanoparticles and protein and aptamer molecules and interact with their counterparts. Based on the detection of these nanoparticles, the amount of the analyte can be determined^[193].

Various nanomaterial labels are used in the sandwich-type immunoassays, including noble metal nanoparticles, semiconductor (quantum dot, QD) nanoparticles, metal oxide nanostructures, carbon nanomaterials, hybrid nanostructures and marker loaded nanocarriers (e.g. silica nanoparticles, apoferritin, and liposome beads)^[194]. Metal, and/or semiconductor nanostructures, i.e. gold and silver nanoparticles as well as quantum dots have been directly used as electroactive labels to amplify the signal in the electrochemical detection of proteins, so this part of the thesis will focus on these labels.

One important goal of using nanoparticle labels in immunoassays is to achieve signal amplification complying with or surpassing that of the enzyme labels (see Table 1), while utilizing more robust and chemically stable molecules enabling new and advanced functions, e.g. the detection of multiple targets. One major advantage lies in the possibility to control and tailor the properties of nanoparticles to meet the needs of specific applications, for example, to provide unique chemical and physical properties (electrical, electrochemical, optical and magnetic)^[193][195]. In particular, nanomaterial labels are showing the greatest promise for developing ultrasensitive electrochemical immunoassays^[196].

Table 1 The number of generated ions and the corresponding amplification effect of gold and silver nanoparticles of various sizes

np diameter Au atoms/np Ag atoms/np Amplification

5 nm 3862 3836 × 10⁴

10 nm 30896 30689

15 nm 104273 103575 × 10⁵

20 nm 247118 245510

25 nm 482750 479512

30 nm 834183 828596 × 10⁶

Quantum dots were initially used as fluorescent biological labels in 1998 (CdSe core with ZnS shell)^[197], and the first use of QD labels for electrochemical monitoring of DNA hybridization was reported in 2002 (CdS)^[198]. The combination of the intrinsic redox properties of QDs with the sensitive electrochemical stripping analysis of the metal components of semiconductor nanoparticles led to very sensitive detection when

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using quantum dot labels in the electrochemical immunoassays, and enabled the simultaneous multiplexed measurement of protein targets utilizing different semiconductor nanoparticle label traces (ZnS, CdS, PbS, CuS)^[199]. Each biorecognition label yielded to a distinct voltammetric peak whose peak potential and current reflected the identity and concentration, respectively, of the corresponding analyte.

The application of colloidal gold/silver as amplification in immunology dates back to the early 1970s when the development of silver-enhanced methods allowed gold labels to provide high-resolution, specific and sensitive immunocytochemistry^[194]. The use of colloidal gold as electrochemical immunoassay label, however, for voltammetric monitoring of protein interactions was pioneered only in 2000^{[200] [201]}. To further enhance the sensitivity of gold nanoparticle label-based immunoassays various techniques have been developed. For example, the nanoparticle-promoted precipitation of silver on gold nanoparticle labels^{[202][203] [204]}, where the silver ions were detected by voltammetric^[202][204] or potentiometric^[203] method after dissolution of the metallic silver. A common problem in Ag enhancement can be the high background signal as a result of non-specific precipitation of silver onto the substrate.

Various techniques have been developed to reduce this non-specific deposition to further increase the sensitivity of this method.