Biosensors Applied to Diagnosis of Infectious Diseases – An Update

Review Article

Austin J Biosens & Bioelectron. 2015; 1(3): 1015.

Biosensors Applied to Diagnosis of Infectious Diseases – An Update

Rodovalho VR1, Alves LM1, Castro ACH1, Madurro JM2, Brito-Madurro AG1 and Santos AR3*

1Institute of Genetics and Biochemistry, Federal University of Uberlandia, Brazil

2Institute of Chemistry, Federal University of Uberlandia, Brazil

3Faculty of Computing, Federal University of Uberlandia, Brazil

*Corresponding author: Santos AR, Faculty of Computing, Federal University of Uberlandia, Avenue Jo&aTilde;o Naves de Ávila 2121, Campus Santa Mônica, Block 1B, Room 120, 38400-902, Brazil

Received: September 26, 2015; Accepted: December 30, 2015; Published: December 31, 2015


In the past five decades, biosensors have consolidated their impact in several fields, including clinical applications, due to advantages such as high selectivity and sensitivity, potential for miniaturization, portability, low cost and rapid response. Recent advances in biomarkers discovery and biotechnology are now clarifying the nuances of many biological processes in health and disease, highlighting new targets for diagnosis and therapeutics. This is especially important in the case of infectious diseases, since the number of predicted deaths remains high, with threats of epidemics and pandemics, emerging and re-emerging diseases and pathogen resistance to antibiotics. Therefore, the availability of robust diagnosis methods is crucial. This review presents the current strategies for diagnosis of infectious diseases, notions about biomarkers and ligand selection, besides focusing on the promising technology of biosensors.

Keywords: Biosensors; Sensors; Biomarkers; Diagnosis; Infectious diseases


ELISA: Enzyme-Linked Immunosorbent Assay; PCR: Polymerase Chain Reaction; MALDI-TOF MS: Matrix-Assisted Laser Desorption/ Ionization Time-Of-Flight Mass Spectrometry; NGS: Next Generation Sequencing; FISH: Fluorescence In Situ Hybridization; SELEX: Exponential Enrichment; SPR: Surface Plasmon Resonance; WHO: World Health Organization; EIS: Electrochemical Impedance Spectroscopy


Infectious diseases are caused by pathogenic microorganisms, including bacteria, viruses, fungi and parasites. Some examples among those enumerated by the World Health Organization (WHO) include tuberculosis, meningococcal meningitis, malaria, AIDS, pneumonia, poliomyelitis, hepatitis, Ebola virus disease, dengue and Chikungunya, American trypanosomiasis (Chagas disease), leprosy, toxoplasmosis and leishmaniasis.

The urbanization process and the consequent lack of city planning, poor management of sanitary conditions and water supplies, great inhabitants’ density and interference in previously untouched ecosystems conjunctly contribute to the spread of infectious diseases [1]. In spite of the existent vaccination programs, the rise in incidence of certain diseases shows the impact of intentional under vaccination and the urge for public health education programs [2], as well as the need of new immunization strategies and alternatives to overcome pathogen resistance to antibiotics [3].

Although the mortality related to infectious diseases is being reduced worldwide, the number of deaths predicted for 2050 is 13 million and the threats of epidemics and pandemics remain considerable [3]. Moreover, emerging and re-emerging diseases caused by new, uncategorized or persistent pathogens have been reported [4].

This review presents the current strategies for diagnosis of some infectious diseases, notions about biomarkers and ligand selection, besides focusing on the promising technology of biosensors.

Diagnosis strategies for infectious diseases

A diagnostic test is any method for identification of a patient’s disease or condition. In the case of infectious diseases, it allows the detection of presence or absence of infection. The importance of simple, accurate, affordable and rapid diagnosis tests is justified by its impact in the clinical management, since early diagnosis affects therapy effectiveness and avoids long-term complications and pathogen transmission [5].

While the standard diagnosis techniques for infectious diseases include well-established methodologies, such as Enzyme- Linked Immunosorbent Assay (ELISA), nucleic acid-based assays, microscopy and microorganism culture [6], the development of new strategies for the evaluation of specific biomarkers in clinical diagnosis is imperative [7]. The diagnosis tests for infectious diseases should present a set of desirable characteristics, such as sensitivity, specificity and reproducibility [5].

Historically, the identification of infectious agents was initially performed by culture and microscopy. Then, antigen detection and Polymerase Chain Reaction (PCR) became widely used. Currently, pathogen identification and host response (e.g., antibodies detection) are both used to diagnosis pathological states [8].

PCR and DNA microarrays are two widely used nucleic acids technologies. PCR employs oligonucleotide primers that are complementary to pathogen genetic material to amplify it, if present in the sample. The reaction product is detected during or after the process. Microarray technology, by the other hand, allows multiple target detection through hybridization with the probes immobilized on a surface [9].

Recently, Matrix-Assisted Laser Desorption/Ionization Time- Of-Flight Mass Spectrometry (MALDI-TOF MS) is being adopted in the comparison of protein fingerprint obtained in a sample with the available databases, for the identification of bacteria, fungi [10] and viral pathogens [11]. There is also the potential of Next Generation Sequencing (NGS) methodologies to revolutionize infectious diseases diagnosis, since it does not rely on pre-established sequence targets, allowing the identification of emerging or mutating pathogens [12,13]. Other strategies are also being developed for clinical diagnosis, including microfluidic [14] and nanotechnological [15] devices.

Biosensors, other promising diagnosis technology that has received attention in the last decades and has several associated advantages will be discussed later in detail.

Biomarkers and their ligands for infectious diseases diagnosis

A biomarker is “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” [16]. Biomarkers are also defined as specific variables, represented by biomolecules, such as genes, proteins and metabolites, associated to certain populations as distinguishable features [17]. Moreover, this term may be associated with the use of genomics, transcriptomics, proteomics and metabolomics technologies, the monitoring of drug discovery, as well as clinical concepts, such as prediction, progression, regression, outcome, diagnosis and therapeutics [18]. Some examples of biomarkers for infectious diseases include C-Reactive Protein (CRP) and soluble Triggering Receptor Expressed on Myeloid cells 1 (sTREM-1). While CRP is applied for sepsis, severe infection, rheumatologic conditions and coronary artery disease risk stratification; sTREM-1 is a sepsis prognostic marker [8].

The ideal biomarkers should present a whole set of desirable characteristics, including accessibility for measurement, sensitivity and specificity. Its clinical importance should be externally validated; its use should result in cost-effective assays [19] and present reproducibility and stability toward sample variations [20].

Biomarkers identification may be achieved by several approaches, including simple statistical tests, development and analysis of classification models or subset-selection optimization [17]. In the last decades, several biomarkers for infectious diseases have been identified, due to improvements in biomolecules screening techniques and bioinformatics analysis. However, their translation into clinical use is still limited [19].

Depending on the class of biomarker, there are several methods for the selection of specific ligands, including phage display and Systematic Evolution of Ligands by Exponential Enrichment (SELEX) [21].

Nucleic acid ligands

Polymerase Chain Reaction (PCR) and Fluorescence In Situ Hybridization (FISH) are two widely used nucleic acid detection technologies. While PCR consists in the in vitro specific DNA amplification, FISH is the fluorochrome-labeling of oligonucleotides for hybridization with the complementary target. Both methods rely on the use of known DNA sequences that relates to specific pathogens [22,23]. For the development of such diagnostic systems based on nucleic acid biomarkers and ligands, the general strategy is to compare sequences in order to find species-specific oligonucleotide targets that are present in high copy numbers in the cells [23].

When designing DNA oligonucleotide probes for several applications, including PCR and DNA hybridization assays, some criteria should be followed: assure their complementarity and specificity for the target, avoid competing secondary structures (dimers and hairpins) and choose adequate melting temperature [24,25].

Most efforts in nucleic acid probes design has been related to PCR [24], FISH [26] and microarray technologies [27]. Cho et al. [28] reported the selection of 23-mer primers for Vibrio cholera detection by quantitative polymerase chain reaction. The primers pair had the outer membrane lipoprotein lolB gene as target and allowed the specific identification of several isolates. Ogura et al. [29] reported a method of microarray probe design based on comparison of edit distance between sequences to avoid cross hybridization with similar probes on the array. Naidoo et al. [30] evaluated Mycobacterium Tuberculosispili (MTP) gene and protein sequences as potential biomarkers for tuberculosis and found they are specific and highly conserved among strains of the Mycobacterium Tuberculosis Complex (MTBC), through BLAST and multi-sequence alignment.

These examples show that DNA-based diagnosis and selection of appropriate biomarkers ligands have focused in comparing sequences. However, this paradigm is changing with the application of Next-Generation Sequencing (NGS), which does not rely on previous knowledge about the pathogens genetic sequence [12].

Protein and peptide ligands

Several techniques are employed for protein biomarker discovery and ligands selection, including mass spectrometry, gel electrophoresis and protein microarrays, the later allowing the study of entire proteomes [31]. Also, peptides microarrays may be employed in the selection of peptide ligands, after computational preselection [32].

Another notable methodology is called phage display and relies on the expression and presentation of a great diversity of peptides or proteins on bacteriophages surfaces, allowing their selection against a target biomolecule [33]. Therefore, this tool can be used to find new reagents for immunological assays, including phage-displayed peptides that mimetize pathogen antigens, with applications like leprosy diagnosis [34] and development of vaccines for visceral leishmaniasis [35]. Wu et al. [36] selected three single-chain variable Fragments (scFv) for detection of Highly Pathogenic Avian Influenza A (HPAI) viruses strains, which could be important to accelerate diagnosis and control outbreaks. Another application is the presentation of antibodies in the bacteriophage surface for potential therapeutic purposes [37], with targets such as the CCR5 HIV coreceptor [38].

Other ligands

Aptamers are high-affinity ligands selected in vitro. Their targets may be specific proteins isoforms or conformations, being analogous to antibodies [39]. Aptamersare selected from libraries through Systematic Evolution of Ligands by Exponential enrichment (SELEX) against several classes of targets, including carbohydrates, proteins and inorganic molecules [40].

Aimaiti et al. [41] selected species-specific aptamers for discrimination of Mycobacterium tuberculosis strains through whole cell SELEX process. Shiratori et al. [42] developed DNA aptamers for proteins of several influenza A virus subtypes and applied them in a successful sandwich detection method. Besides diagnostic applications, aptamers have also been used as therapeutics agents, such as the recently reported S15 aptamer, that binds to the envelope protein of all dengue serotypes, neutralizing the infections [43].

These examples show how the integration of physiology, biochemistry, genetics, bioinformatics and other research fields, may improve biomarker discovery and selection of specific ligands, allowing a better understanding of infectious diseases and the development of effective diagnostic systems, such as biosensors [44].

Biosensors for diagnosis of infectious diseases

Biosensors are analytical devices that convert a biochemical recognition event into a measurable signal [45], consisting mainly of a probe (biological recognition element) and a physicochemical detector (transducer) (Figure 1). The objective is to determine the presence, activity or concentration of an analyte in a solution [46], with a broad range of applications, such as industrial [47], environmental [48] and medical [49,50].