**3. Aptamers**

Both macroarrays and microarrays can have two application forms for the DNA microarray technology: the identification of sequence (gene/gene mutation) and determination of ex‐

The determination of expression level of genes the microarrays can study the transcriptome or the proteome. For the transcriptome microarrays the probes consist on cDNA that hybrid‐ ize with the mRNA of the cell. By the other hand, the proteome microarrays can use proteins as probe or the antibody making the antigen-antibody reaction. One example of this micro‐ array is the peptide microarray analysis of *in silico*-predicted epitopes for serological diagno‐

About the identification of gene sequence, microarray should have genomic DNA as probe of a specific chromosome, specifically all the genes that compose the chromosome. Or when a microarray only has a gene with one or more different nucleotides called Single Nucleo‐

These microarrays are used to determinate the cancer progression; all the changes on these gene are important to establish a clinical forecast [20]. Such microarrays have been used for the detection of specific bacteria [22, 23], species determination [24], and screening of envi‐

Chin-I *et al.,* coupled 16S rDNA PCR and DNA hybridization technology to construct a microar‐ ray for simultaneous detection and discrimination of eight fish pathogens (*Aeromonas hydrophi‐ la, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae* and *Vibrio anguillarum*) commonly encountered in aquaculture. The array comprised short oligonucleotide probes complementary to the poly‐ morphic regions of 16S rRNA genes from the target pathogens. The results showed that each

Yu-Cheng *et al.,* designed the DNA probes and PCR primers for the detection of *Listeria mon‐ ocytogens, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli* O157:H7*, Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae*and *Pseudomonas fluorescens* by using two sets of multiplex PCR, followed by a chromogenic macroarray system, these organisms in

An example of microarray designed for infection diagnosis is a microarray developed by Uchida *et al.,* for the direct detection of pathogens in osteoarticular infections by polymerase

And finally, and the most interesting DNA microarray used for the endophthalmitis diagno‐ sis is the one developed by Tsutomu *et al.* They used 13 samples of vitreous fluid (VF) ob‐ tained from 13 patients during vitrectomy. Vitreous fluids from three patients with suspected endophthalmitis and ten controls without infection were subjected to testing for the presence of bacteria and fungi in culture tests, polymerase chain reaction (PCR) analysis, and DNA microarray analysis. The DNA microarray contained the spots for 16S rDNA, var‐ iable and conserved areas for bacteria, and the 18S rDNA for fungi. No control sample was positive for bacteria or fungi in the culture test, PCR, or microarray analysis. Specimens from two patients (Cases 1 and 2) with suspected endophthalmitis were positive for bacteria

ronmental sequences related to a certain function within a community [25, 26].

probe consistently identified its corresponding target strain with 100% specificity [27].

milk or other food products could be simultaneously detected [28].

chain reaction amplification and microarray hybridization [29].

pression level of genes.

6 Common Eye Infections

sis of *Toxoplasma gondii* infection in humans [19].

tide Polymorphism (SNP) can detect a gene mutation [20].

The term aptamers derives from the Latin *aptus*, it means to adapt [31]. Aptamers are syn‐ thetic nucleic acids (DNA or RNA) that bind specifically to a wide variety of molecules in‐ cluding metal ions K2+, Hg2+, Pb2+, ATP, antibiotics, amino acids, vitamins, organic dyes, peptides and proteins, additionally aptamers are not immunogenic and non-toxic, superior to antibodies [32, 33, 34, 35]. Thirty and 60 nucleotides usually comprise the length of the central region, so that the total length of the aptamer is 70 to 100 nucleotides. For selection of aptamers with higher affinity the SELEX method is used (Systematic Evolution of Ligands by Exponential Enrichment). Wherein the target molecules are incubated with a population of aptamers, which interact with the target molecule by affinity, non-interacting target mole‐ cules are removed and the oligonucleotides are amplified by PCR and characterized by se‐ quencing, being able to maintain a stock by its introduction to bacteria using plasmids. After obtaining the individual aptamers were characterized by their interaction with the target molecule by techniques as SPR, ELISA, Western blot or slot blot [36].

#### **3.1. Applications aptamers**

The aptamers can be used in different areas of study; some of its applications are reviewed below.

Biotechnology: the aptamers can be used for protein purification [37] and also for the devel‐ opment of techniques such as western blot or chromatin immunoprecipitation [38], also to monitor the status of phosphorylation of proteins *in vivo* [35]. There is an aptamer with ac‐ tivity of inhibitor of coagulation factor IXa by the addition of antisense RNA, this is an im‐ portant method to control bleeding in patients who are intolerant of heparin, the aptamer is of interest for therapeutic and diagnostic [40].

Therapy: The therapeutic targets can be divided into two classes, the intracellular targets such as transcription factors, and extracellular targets such as viruses. The aptamers can be administered intravenously or subcutaneously; there is also topical application to prevent pathogens interaction with their receptors on mucosal surfaces. The release of intracelular‐ aptamers to bind their targets has been made mainly by the incorporation into liposomes or by systems of viral-based expression. A technique using a liposome to release viral vector fusigenicaptamer DNA in target cells, showed sequestration of E2F (transcription factor) leading to a reduction in the growth of abnormal vascular tissue that is typically seen after angioplasty [41]. Some research groups have studied the expression of aptamers in cells. An example of this is the expression of a chimeric transcript initiating sequence, consisting of a human tRNA-Met and anti-HIV reverse transcriptase-pseudoknotaptamer under control of RNA polymerase III promoter in human 293 cells. The chimeric RNA resulted in a reduction of over 75% in viral replication, similar results were observed when carrying out the trans‐ fection in Jurkat cells [41]. The FDA (Food and Drug Administration, USA) approved the system Eyetech /Pfizer's aptamer (Macugen) for treatment of related macular degeneration. The target of Macugen is VEGF (Vascular Endothelial Growth Factor), preventing choroidal neovascularization [43, 44]. There are aptamers against amyloidogenic proteins such as pep‐ tide Aß associated with Alzheimer disease [45] and against abnormal proteins in prion dis‐ easesand scrapie, and Creutzfeldt-Jakob disease [46, 47].

#### **3.2. Diagnostics and biosensors**

The high affinity and specificity of aptamers make them ideal as reagents for diagnosis. And al‐ so aptamers can be detected by differential staining fluorescence that results in a high sensitivi‐ ty. There are an aptamers called ''beacons'' that have many uses ranging from detection of environmental pollutants and thus also to monitor the levels of carcinogens or drugs in the blood [48]. The development of quantum dot aptamers also could help to establish the role of aptamers as biosensors [49, 50]. The quantum dots are novel fluorophores with a different emis‐ sion profile, but all they are excited in the same wavelength. In this system multiple copies of an aptamer is attached to a single quantum dot, and each aptamer base is binding to a complemen‐ tary strand. The plug moves on ligand binding, leading to large increases in fluorescence emis‐ sion. If different aptamers are immobilized each on a single quantum dot, multiple ligands can be detected in a single assay. The aptamers have great potential as early warning systems to de‐ tect cell surface binding to damaged or diseased cells.

#### **3.3. Aptamers: An approach to diagnostic microbiology**

It has previously been addressed different approaches to the application of aptamers. The use of aptamers in microbiology is interesting, in order to have new tools for the diagnosis of infections. Today, several research groups are involved in aptamers development aimed at the detection of microorganisms. Duan *et al.,* by means of the system evolution of ligands by exponential enrichment (SELEX) developed a DNA aptamer labeled with carboxyfluores‐ cein (FAM) that binds specifically to *Vibrio parahaemolyticus* [51]; Zelada *et al.,* through ap‐ tamer system potentiometric biosensors based on carbon nanotubes attached to a single wall (SWCNT) were able to identify and detect *Escherichia coli* with a linear response [52]. Aptam‐ ers represent a very flexible technique for the detection of microorganisms such is the case of the determination of *E. coli* based on immunomagnetic separation and real time PCR ap‐ tamers, this technique consists of three steps, first the binding of *E. coli* to an antibody conju‐ gated to a magnetic bead, the second RNA aptamer is captured on the surface of *E. coli* forming a sandwich and then a heat process release the aptamers and these are amplified using real time PCR. The sensitivity of this method allowed the detection of 10 *E. coli* in 1 mL of sample [53]. Aptamers have also been developed for quantum dot fluorescence assays against *Bacillus thuringiensis*, detecting up to 1000 CFU/ml [54]. Application of aptamers in the microbiological diagnosis and the advantages respect to other diagnostic techniques must be analyzed; however there is not much information about the application of aptamers in the microbiological field.

#### **3.4. SOMAmers**

leading to a reduction in the growth of abnormal vascular tissue that is typically seen after angioplasty [41]. Some research groups have studied the expression of aptamers in cells. An example of this is the expression of a chimeric transcript initiating sequence, consisting of a human tRNA-Met and anti-HIV reverse transcriptase-pseudoknotaptamer under control of RNA polymerase III promoter in human 293 cells. The chimeric RNA resulted in a reduction of over 75% in viral replication, similar results were observed when carrying out the trans‐ fection in Jurkat cells [41]. The FDA (Food and Drug Administration, USA) approved the system Eyetech /Pfizer's aptamer (Macugen) for treatment of related macular degeneration. The target of Macugen is VEGF (Vascular Endothelial Growth Factor), preventing choroidal neovascularization [43, 44]. There are aptamers against amyloidogenic proteins such as pep‐ tide Aß associated with Alzheimer disease [45] and against abnormal proteins in prion dis‐

The high affinity and specificity of aptamers make them ideal as reagents for diagnosis. And al‐ so aptamers can be detected by differential staining fluorescence that results in a high sensitivi‐ ty. There are an aptamers called ''beacons'' that have many uses ranging from detection of environmental pollutants and thus also to monitor the levels of carcinogens or drugs in the blood [48]. The development of quantum dot aptamers also could help to establish the role of aptamers as biosensors [49, 50]. The quantum dots are novel fluorophores with a different emis‐ sion profile, but all they are excited in the same wavelength. In this system multiple copies of an aptamer is attached to a single quantum dot, and each aptamer base is binding to a complemen‐ tary strand. The plug moves on ligand binding, leading to large increases in fluorescence emis‐ sion. If different aptamers are immobilized each on a single quantum dot, multiple ligands can be detected in a single assay. The aptamers have great potential as early warning systems to de‐

It has previously been addressed different approaches to the application of aptamers. The use of aptamers in microbiology is interesting, in order to have new tools for the diagnosis of infections. Today, several research groups are involved in aptamers development aimed at the detection of microorganisms. Duan *et al.,* by means of the system evolution of ligands by exponential enrichment (SELEX) developed a DNA aptamer labeled with carboxyfluores‐ cein (FAM) that binds specifically to *Vibrio parahaemolyticus* [51]; Zelada *et al.,* through ap‐ tamer system potentiometric biosensors based on carbon nanotubes attached to a single wall (SWCNT) were able to identify and detect *Escherichia coli* with a linear response [52]. Aptam‐ ers represent a very flexible technique for the detection of microorganisms such is the case of the determination of *E. coli* based on immunomagnetic separation and real time PCR ap‐ tamers, this technique consists of three steps, first the binding of *E. coli* to an antibody conju‐ gated to a magnetic bead, the second RNA aptamer is captured on the surface of *E. coli* forming a sandwich and then a heat process release the aptamers and these are amplified using real time PCR. The sensitivity of this method allowed the detection of 10 *E. coli* in 1 mL of sample [53]. Aptamers have also been developed for quantum dot fluorescence assays

easesand scrapie, and Creutzfeldt-Jakob disease [46, 47].

tect cell surface binding to damaged or diseased cells.

**3.3. Aptamers: An approach to diagnostic microbiology**

**3.2. Diagnostics and biosensors**

8 Common Eye Infections

SOMAmers (Slow Off-rate Modified Aptamers) are single-stranded deoxynucleotides type aptamers selected *in vitro* from large random libraries, for their ability to bind small mole‐ cules, peptides or proteins [55, 56]. SOMAmers are aptamers carrying dU residues in posi‐ tion 5 that are involved in interactions with target molecules [57].

SOMAmers have been created for more than 1000 protein targets of different molecular functions, including known diseases and physiological associations. The target families broadly include receptors, kinases, growth factors and hormones, and also include a diverse array of intracellular and extracellular proteins.

The core of the reagents is a SOMAmer coupled to biotin, via a photocleaveable linker al‐ lowing binding to streptavidin of the complex in the washing steps (Figure 2). A fluoro‐ phore Cy3 incorporated into the capture reagents allows quantification of protein available commercially available systems based microarray systems but not necessary for all assay formats (SomaLogic ®).

**Figure 2.** SOMAmer-protein complex. SOMAmer binds specifically to protein target through interacting motifs. Func‐ tional groups are: B= biotin for capture; F=Cy3 for detection and L=photocleavable linker.

#### *3.4.1. SOMAmers applications*

Comparison between proteome of healthy and diseased tissues from human using Somas‐ can, can provide major knowledge of the biology of the disease and may lead to the discov‐ ery of new highly specific biomarkers for diagnosis, prognosis and therapeutic targets for the development of new drug treatment and it will improve personalized medicine.

Somascan premium has been used for the discovery of biomarkers for the detection of meso‐ thelioma in the population exposed to asbestos. SOMAmers reagent showed better perform‐ ance with respect to the ELISA test. Also the system is used to discover biomarkers for the detection of non-small cell lung cancer [57]. Moreover the system can be applied in tumor tissue lysate to obtain biomarkers associated with the disease as well SOMAmers same re‐ agents can be used for histochemical evidence [58]. The SOMAmers represents an effective tool for biomarker discovery in different areas such as oncology, neurology, cardiovascular and metabolic diseases. To microbiological purposes as those related to the detection of agents in microbial infections SOMAmers represent a good alternative tool that may be ap‐ plied for microbiological diagnosis in the future.
