Immunosensors reported in scientific journals in the last decade
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Brewer completed her undergraduate degrees at Stanford University, with a B.S. with Honors in Biological Sciences and an A.B. in Comparative Literature with interdisciplinary Honors in Humanities. She continued on at Stanford in a dual-degree graduate program, graduating with an M.D. and a Ph.D. in Neuroscience in 2007. Her work in graduate school with Brian Wandell, Ph.D., focused on computational neuroimaging measurements of visual cortex organization and plasticity in humans and macaque. She now is an Associate Professor in the Departments of Cognitive Sciences and Language Science, by courtesy, at the University of California, Irvine. Dr. Brewer’s research focuses on visual, auditory, and multi-sensory neuroscience, using behavioral, genetic, and high-resolution neuroimaging techniques to investigate questions ranging from the fundamental organization of human visual and auditory cortex to plasticity in visual, auditory, and sensorimotor regions.",institutionString:"University of California, Irvine",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"6",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"University of California, Irvine",institutionURL:null,country:{name:"United States of America"}}}],coeditorOne:{id:"149246",title:"Dr.",name:"Brian",middleName:null,surname:"Barton",slug:"brian-barton",fullName:"Brian Barton",profilePictureURL:"https://mts.intechopen.com/storage/users/149246/images/system/149246.jpeg",biography:"Dr. Brian Barton received his B.S. in Psychology at the University of Oregon, where he studied visual attention and working memory with Edward Awh, Ph.D., and Edward Vogel, Ph.D. Dr. Barton then earned his Ph.D. in Psychology with a Concentration in Cognitive Neuroscience in 2013 at the University of California, Irvine (UCI), where he expanded his research into functional MRI measurements of visual cortex organization and plasticity with Alyssa A. Brewer, M.D., Ph.D. He continued on at UCI as a post-doctoral scholar for three years with Drs. Brewer, Greg Hickok, Ph.D., and Kourosh Saberi, Ph.D., applying his vision work to measurements of the organization of human auditory cortex. 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They form one of the most important classes of affinity biosensors based on the specific molecular recognition of antigens by antibodies to form a stable complex, in a similar way to immunoassay. In contrast to immunoassay, modern transducer technology enables the label-free detection and quantification of the immune complex [1]. The immune system possesses a great ability to distinguish self from non-self. Antibodies (Abs) with high specificity are synthesized by the organism in reasonable quantities after sensing the foreign species called antigens (Ag), as part of the immune defence system. The ability of organisms to recognize the presence of Ags and to respond rapidly by synthesizing Abs that exhibit high binding constants [2] was and continues to be exploited by scientists in order to create new specific analytical devices. Molecules generally designed as Abs include a number of classes and subclasses of immunoglobulins with high specificity towards various targets.
The high sensitivity and selectivity of the immunoreactions together with the availability of Abs or Apt for a broad range of molecules make immunochemical methods useful tools in a large variety of applications, including clinical analysis. The use of immunosensors instead of other immunochemical techniques simplifies the analysis considerably, making it rapid and reliable [3].
Lately, a plethora of biomolecules whose presence or level of expression is an indicator of some pathological condition (we refer here to biomarkers) are currently used in immunoassays. Those devices represent a convenient way of measuring the concentration of biomolecules in biological fluids (such as serum, urine, etc.) by means of immunological reactions.
Most clinical protein biomarker detection is done today by enzyme-linked immunosorbent assay (ELISA), but requirements for the relatively expensive test kits and the plate readers limit ELISA’s usefulness for fast diagnostics [4]. LC-MS-based proteomics is currently used for biomarker discovery, but is currently too expensive and technically too complex for routine clinical diagnostics [5, 6]. Microarrays developed on a 96- or 384-well plate format, Ab-coated with colorimetric detection are also being used in clinical diagnostic. These arrays are simple and highly selective and allow multiplexed measurement of proteins [7, 8, 9, 10]. Several commercially available automatic or semiautomatic analysers for multiplexed protein measurements, employing fluorescence (Luminex, Myriad RBM), electrochemiluminescence (ECL) (Roche Diagnostics, Mesoscale Discovery), or surface plasmon resonance (Horiba Inc., BIO-RAD; Biacore Life Sciences, GE Healthcare) measurement technologies are currently used in hospital laboratories [11]. Assay kits are available for measurement of up to 10 selected target proteins per sample with detection limits (LOD) of 1-100 pg/mL-1 in serum [4]. These commercial instruments require specialized consumables, including sample well plates, chips and reagent kits [12], which are expensive and, thus, they have limited usefulness for point-of-care (POC) applications where resources are limited. Fluorescence-based detection strategies typically require laser sources and precise alignment of optical components, whereas electrochemical detection strategies offer robust, quantitative measurements using low-cost, simple instrumentation [11].
Depending on the type of
The role of electrochemical immunosensors in analysis has considerably increased in the last years, due to their low cost, high sensitivity, potential for miniaturization and automation, and low power requirements. Important developments have been secured, especially in sensor design concerning the type of bioelement (Ab, Apt, Affibody, miRNA) and the immobilization technique.
The immunoassay is a device based on biochemical detection and measurement of biomolecules in a test solution using a specific Ab or immunoglobulin. Immunoassays rely on the specificity of the immunoreaction, and more precisely on the ability of an Ab or Apt to recognize and bind to a specific target, usually an Ag. The measurable signal proportional to the Ag concentrations is obtained with the aid of a label. Some of the commonly employed labels include radioisotopes [16], chromophores [17], fluorophores [18] and enzymes [19]. Depending on the nature of the label, the immunoassay could be classified into radioimmunoassay, chromoimmunoassay, fluoroimmunoassay and enzyme immunoassay, respectively [20].
The enzyme immunoassay or enzyme-linked immunosorbent assay (ELISA) is the most commonly used technique employed as a diagnostic tool for clinical purposes. The working principle of ELISA consists in the immobilization of an unknown amount of Ag onto a solid surface, and then a specific Ab is added over the surface so that it can bind to the Ag. This antibody is usually labelled with an enzyme; the substrate of the enzyme is then added, which will be converted into a compound that can be quantified by means of optical methods. There are four types of ELISA: direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA [20].
The simplest protocol is the one used in the case of direct ELISA [21]. The Ag is adsorbed to a plastic plate, then an excess of another protein (normally bovine serum albumin) is added to block the free binding sites. An enzyme is linked to an Ab in a separate reaction and the formed enzyme-Ab complex is added to the well. The Ab interacts with its Ag, forming an Ag/Ab-enzyme complex, and the excess of unreacted Ab-enzyme is washed off. Afterwards the substrate of the enzyme is added, and is converted into a compound that gives a measurable signal directly linked to the Ag quantity.
Indirect ELISA is a two-step ELISA assay which involves the use of two Abs to bind the Ag: a primary capture Ab and a secondary signalling Ab. The primary Ab is incubated with the Ag followed by the incubation with the secondary Ab. However, this may lead to non-specific signals because of a cross-reaction that the secondary Ab may bring about. This technique has a high sensitivity because more than one labelled Ab is bound per Ag molecule.
Sandwich ELISA is a less common alternative, but is highly efficient in sample Ag detection. Moreover, many commercial ELISA pair sets are built on this sandwich ELISA.
The sandwich ELISA quantifies antigens between two layers of antibodies (i.e., capture and detection antibody). The Ag to be measured must contain at least two antigenic epitopes capable of binding to Ab, since at least two Abs act in the sandwich. Either monoclonal or polyclonal Abs can be used as the capture and detection Abs in sandwich ELISA systems. Monoclonal Abs recognize a single epitope that allows fine detection and quantification of small differences in Ag. A polyclonal is often used as the capture Ab to pull down as much of the Ag as possible. The advantage of sandwich ELISA is that the sample does not have to be purified before analysis, and the assay can be very sensitive (up to two to five times more sensitive than direct or indirect ELISA, but lower than ELISpot). The central event of competitive ELISA is a competitive binding process executed by original Ag (sample Ag) and add-in Ag. The procedures of competitive ELISA are different in some aspects compared with indirect, sandwich and direct ELISA assays. In competitive ELISA assays, the following successive steps are involved: 1) primary Ab (unlabelled) is incubated with sample Ag; 2) Abs-Ag complexes are then added to 96-well plates which are pre-coated with the same Ag; 3) unbound Ab is removed by washing the plate (the more Ags in the sample, the fewer Abs will be able to bind to the Ag in the well, hence "competition"); 4) the secondary Ab that is specific to the primary Ab and conjugated with an enzyme is added; 5) a substrate is added, and the remaining enzymes elicit a chromogenic or fluorescent signal. For competitive ELISA, the higher the sample Ag concentration, the weaker the eventual signal is. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any Ag that may be present.
In order to avoid confusion, the term ‘immunoassay’ is used for tests based on immunoreactions, while the term ‘immunosensor’ is specifically employed to describe whole instruments, i.e., immunoreaction-based biosensors. In the heterogeneous immunoassays, the Ab or Ag is immobilized at a solid substrate (e.g., microplate), while homogeneous immunoassays take place in solution [22]. Heterogeneous immunoassays are easier to design and construct, but they require a separation of the Ag and the Ab from the samples and their immobilization at a solid surface. In contrast, the homogeneous immunoassays usually involve the immobilization of the biomolecules on nano-/microbeads, thus allowing the integration of multiple liquid handling processes [23, 24]. The homogeneous immunoassays coupled with microfluidic devices are relatively inexpensive and efficient alternatives and can be used for the detection of complex samples, such as urine or blood, without any sample pre-treatment or large volume consumption. Microfluidic lab-on-a-chip technology also has the advantages of portability, integration and automation.
In the immunosensor design, the sensing element is formed by means of immobilization of Ags or Abs, and the binding event is transformed into a measurable signal by the transducer [25]. The performances of immunosensors are obviously related to the selectivity and affinity of the Ab-analyte binding reaction. The immunosensors can be either direct (where the immunochemical reaction is directly determined by measuring the physical changes induced by the formation of the complex) or indirect (where a sensitively detectable label is combined with the Ab or Ag of interest, Figure 1) [20]. The distinction has an entirely different meaning to the terms direct and indirect in the immunoassay fields, which are both tracer-related. They are distinguished by whether Ab binding is directly detected, for example, after an enzyme-labelled Ab is bound to an immobilized coating conjugate or an enzyme tracer to an immobilized Ab, or whether the detection only takes place after a secondary binding reaction, for example, if an enzyme-labelled secondary Ab is used to label the first Ab bound to an immobilized coating conjugate [21, 22, 23].
Scheme of the sandwich assay for protein biomarker detection using magnetic beads
Immunoassays can be performed either in competitive or sandwich-type configurations. The competitive-type assay is based on the competition between the analyte in the sample which is unlabelled and a labelled analyte for a limited number of captured Abs. Two approaches can be employed: the first is based on immobilized Abs that react with free Ags in competition with labelled Ags; the second consists in immobilized Ags and labelled Abs. The sandwich-type ELISA-like assay is the most widely used format and employs a pair of matched, high-affinity Abs and appropriate labels, leading to an increased sensitivity and selectivity. The Ag of interest is captured between a pair of Abs and a labelled Ab is further used to obtain a detectable signal [26, 27, 28].
The reaction of proteins in solution with their complementary proteins immobilized on a surface leads to changes in, for example, refractive index, thickness and dielectric constant of the immobilized layer. With proteins immobilized on a piezoelectric material, a change in resonance frequency is detected which is proportional to the mass change on the surface. These properties are exploited in optical, electrochemical and piezoelectric immunosensors. Ideally, immunosensors are devices with fast response, a high specificity and sensitivity. It is also preferable that they are regenerable, which means that they can be reused immediately after dissociation of the Ab-Ag complex, e.g., by using a chaotropic reagent [29].
To be able to detect the interaction, one of the immunoagents has to be labelled. Various labels have been applied, of which radioisotopes were among the first because of their inherent sensitivity. Other frequently used labels are chemiluminescent compounds and enzymes (e.g., alkaline phosphatase, horseradish peroxidase), which convert an enzyme substrate into a measurable product (Figure 2).
Bioconjugates for signal amplification strategies: enzymes, nanoparticles, multienzyme nanoparticles, multienzyme CNTs (carbon nanotubes), nanoparticle QDs (quantum dots)
Many different configurations have been described and the four most commonly used formats are shown in Figure 3. In a homogeneous immunoassay Abs (Figure 3a), Ags and labelled Ags are mixed. Free labelled Ags and those bound to an Ab, can be distinguished by a change of the label’s activity upon binding. Usually, immunoassays are heterogeneous, which means that either the Ab or the Ag is immobilized on a solid carrier and an immunocomplex is formed upon contact with a solution containing the other immunoagent.
(a) Homogeneous competitive immunoassay; (b) Heterogeneous non-competitive immunoassay; (c) Heterogeneous competitive immunoassay; (d) Heterogeneous competitive immunometric assay
The unbound proteins are removed by washing and the response obtained from the labels is proportional to the amount of protein bound. In a sandwich immunoassay, Abs are immobilized and after addition of the sample containing the Ag, a labelled secondary Ab is added (Figure 3b). Besides these non-competitive formats, competitive configurations can also be applied. In a competitive assay, competition takes place between free and bound Ag for a limited amount of labelled Ab (Figure 3c) or between Ag (the sample) and labelled Ag for a limited amount of Ab (Figure 3d) [30]. Electrochemical detection of immune interaction can be performed both with and without labelling [31]. A frequently used format in electrochemical immunosensing is the amperometric immunosensor, where proteins are labelled with enzymes producing an electroactive product from an added substrate [32]. Direct detection without labelling can be performed by cyclic voltammetry, chronoamperometry, impedimetry, and by measuring the current during potential pulses (pulsed amperometric detection). These methods are able to detect the electrode change in capacitance and/or resistance, induced by the binding of protein. These immunosensors have been developed using various supporting materials. Those built on silicon, silanized metal, or polypyrrole are often regenerable, while those based on SAMs, on silver or gold, are not. Chaotropic reagents, used to accomplish disruption of the Ab-Ag bond, have been reported to affect the SAM [33, 34] or to induce a decrease in the sensitivity of the sensor [34, 35].
The most common bioelements used in the development of electrochemical immunosensors are Abs, followed by Apt, and in the last five years the miRNA. In order to perform an early diagnosis, a method that is able to measure peptides and proteins
Abs are proteins produced in animals by an immunological response to the presence of a foreign substance (with a molecular weight larger than 1.5 kDa), a so-called Ag, and which have a specific affinity for this Ag [36, 37, 38, 39, 40]. Abs may be monoclonal or polyclonal. Polyclonal Abs have a specific affinity for the Ag, but they are directed to different binding sites (epitopes) on the Ag with different affinities. In immunoassays, often the use of monoclonal Abs is preferred. These Abs are formed by using identical cells cultivated by the hybridoma technology, and have optimized characteristics such as high affinity or high specificity. To conclude, polyclonal Abs are isolated after immunization of animals, while monoclonal Abs require hybridomal selection. Abs are structurally very similar. Of the five classes of immunoglobulins (IgA, IgD, IgE, IgG and IgM), which differ in glycosylation and the number and position of disulphide bridges, IgG (150 kDa) are mainly used for immunoassays. An IgG consists of two heavy and two light chains, which are interconnected by disulphide bridges (Figure 4). All chains have a variable and a constant region. The variable regions of the heavy and light chain combine in one interaction site for the Ag, which is called the antigenic site. Thus, an IgG molecule has two identical binding locations for the Ag [41, 42]. Abs often demonstrate strong binding features and high selectivity; however they have some intrinsic limitations related to their molecular properties. The most commonly used Ab type, the IgG molecule, is a large, bivalent, multidomain protein, dependent on disulphide bonds and with a complex glycosylation pattern. This explains its relatively poor heat stability and a difficult and expensive manufacturing process. In addition, Abs use only a minor part of the molecule for Ag recognition [43].
A number of techniques are known today for the
Affibody molecules are a class of engineered affinity proteins with high potential for therapeutic, diagnostic and biotechnological applications. Affibody molecules are small (6.5 kDa), single-domain proteins that can be isolated for high affinity and specificity to any given protein target [43].
(A) Illustration of the structure of a typical monoclonal Ab along with the various sites of heterogeneity that are commonly observed [
Affibody molecules have already been investigated as affinity probes in ELISA setup and protein microarrays. For example, in one such study, dimeric Affibody molecules with affinity for IgA, IgE, IgG, TNFα, Insulin and Taq polymerase were immobilized on thiol dextran microarray and an LOD of 70 fM was obtained [44]. Affibody molecules were also evaluated as capture agents in a sandwich array format with unlabelled target protein and monoclonal Abs for detection, demonstrating specificity in a complex serum or plasma sample. The use of Affibody molecules in sandwich assays for analysis of human samples might also be advantageous in a cross-reactivity perspective, since human anti-animal Abs are occasionally present which might lead to cross-linking of capture and detection Abs, resulting in elevated background signals, which are avoided when affibodies are used [44].
Apts, derived from the Latin word “aptus” (meaning to fit) are synthetic single-stranded DNA or RNA molecules that can bind with high affinity and specificity to their target molecules. These artificial “nucleic acid” ligands can be generated against proteins, small molecules, ions, whole cells, tissues and organs [45]. Those specific nucleic acids, selected from random sequence pools, have been shown to bind various targets that do not have a nucleic acid structure. The development of
It was demonstrated that the Apts could bind their targets by adaptive binding. Thus, small molecules usually become an integral part of a three-dimensional structure, while for proteins, the oligonucleotide combines with the protein into a larger molecular structure [46].
Apts offer several advantages over Abs. Being isolated through an
Discovered in 1993 [49], microRNAs (miRNAs) are 22 nucleotide-long non-coding RNA molecules that regulate the expression of target genes at the post-transcriptional level by either translational repression or degradation of miRNAs [50]. It has been estimated that the human genome may encode over 1000 miRNAs, which are thought to regulate about 60 % of the mammalian genes. Altered expression patterns of miRNAs have been associated with many diseases, including several types of cancer, cardiovascular disorders, diabetes, rheumatic disease and neurological disorders [51]. Due to their unique miRNA expression signatures that have been demonstrated in many pathological conditions, these molecules have been proposed as novel biomarkers in diagnostics. The miRNA profiles in clinical samples will not only signify the presence of a disease, but can also help in the prediction of its course and help to select personalized treatments. miRNAs are currently detected in blood serum, plasma, urine, saliva, sweat and other bodily fluids. The levels of circulating miRNAs in serum were estimated to be in the 200 aM to 20 pM range [52]. The field of circulating miRNA analysis is in its early stages, but it has great potential due to the fact that miRNAs are relatively stable molecules and can be easily accessible in a non-invasive manner.
There is a strong and continuous demand for fast, easy and cheap analytical devices for trace detection of compounds of clinical and environmental interest, as well as for pharmaceutical and food industries. The high selectivity and sensitivity of immunosensors make them the most appropriate for the detection and monitoring of the above mentioned domains. The recent developments made in the immunosensor field regarding the optimal orientation of Abs, incorporation of nanomaterials for increased sensitivity, multiplexing and microfluidic-based devices may have potential for promising use in industry and clinical analysis.
Some examples of assays for several commercially available biomarkers are presented in Table 1.
Immunosensors reported in scientific journals in the last decade
The most important feature of immunosensors, the specificity of Ab-Ag reactions, makes them attractive candidates for developing diagnostic tools. Other advantages immunosensors may offer are: decreased medical cost by reducing, e.g., the hospitalization period due to their rapid analysis response; ease of use and miniaturization, making them suitable for “in-home” or “POC” use, thus improving the compliance of patients in follow-up therapy; and rapid response, which can have a determining role in life-threatening diseases such as myocardial infarct or infectious diseases. Different molecules such as proteins, nucleic acids, metabolites, hormones, whole cells and various pathogens are bioanalytical targets for the development of diagnostic devices.
The high prevalence of cancer worldwide, together with its high recurrence and high mortality, creates an urge for rapid, reliable and sensitive detection methods, focusing the scientists’ attention on proteins that are frequently investigated as cancer biomarkers. Cancer biomarkers, like all other biomarkers, are generally defined as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention” [97].
Their aberrant expression in biological fluids (serum, saliva) or cells and tissues can be an indicator of the presence of neoplastic processes. Because their abnormal levels appear from the first stages of cancer, they can play an important role in early stage detection, improving the survival rate of patients. Generally, more than one cancer biomarker is overexpressed for the same type of cancer. Therefore, in order to avoid false positive results, a panel for simultaneous detection of several significant cancer biomarkers is usually required.
Several strategies will be presented below for the detection of cancer protein biomarkers, such as Mucin 1 (MUC1, CA 15-3), prostate-specific antigen (PSA), cancer antigen CA125 and carcinoembryonic antigen (CEA), with an emphasis on electrochemical immunosensors reported in the last years.
Mucins are glycoproteins, either secreted or membrane bound, that have a specific pattern of expression altered during tumour progression, recently investigated as new potent biomarkers in epithelial cancers [98]. Among them, MUC1 has been more extensively studied, being involved in tumour proliferation and metastasis [99], as well as in chemoresistance and alteration of drug metabolism [100, 101]. Overexpression of MUC1 has been associated with breast, ovarian, pancreatic, liver, colon or lung cancer.
An Ab-lectin sandwich assay coupled with optical detection was proposed by Park
Preparation of sandwich aptasensor for MUC1 detection: (a) o-ABA polymerization; (b, c) MUC1 monoclonal mouse Ab immobilization onto PABA-modified graphite SPEs by carboxyl activation; (d) PABA free binding sites blocking with 10 mM ethanolamine; (e) incubation with human MUC1 of different concentrations; (f) specific binding of Apt with human MUC1 Ag (g) interaction of MB with G bases in Apt; (h) DPV measurements [
After the reaction with Ag, the Apt binds to the immunocomplex which allows the accumulation of MB by interaction with the guanine bases of the Apt. DPV was then performed to measure the oxidation peak of MB. A linear response of the current with MUC1 concentration was observed in the range of 1-12 ng/mL with an LOD of 0.62 ppb [102]. Other strategies for MUC1 detection include electrochemical aptasensors based on MB-labelled hairpin Apt, where the detection is based on the conformational changes of the labelled Apt after the binding of the protein [103] or on a dual signal amplification strategy, using poly(
PSA is produced by the prostate gland with normal levels ranging from 0 to 4 ng/mL, and it is overexpressed in prostate cancer. A label-free immunosensor based on white light reflectance spectroscopy for the determination of total- and free-PSA in human serum was reported. A two-site immunoassay was used, employing a biotinylated Ab in combination with streptavidin. High sensitivity was achieved with LOD of 0.2 and 0.15 ng/mL for total and free PSA, in the range of 0.5 to 100 ng/mL, and 0.5 to 20 ng/mL for total and free PSA, respectively [56]. Another interesting approach was used for the construction of a 3D label-free immunosensor for PSA-based crumpled graphene-gold nanocomposites (Figure 6). The graphene sheets decorated with AuNPs were prepared by aerosol spray pyrolysis. LOD of 0.59 ng/mL and a linear range of 0-10 ng/mL were achieved, due to the synergistic effect of graphene and Au, with increased biocompatibility, high level of bound Ag and fast electron transfer. The sensors also proved to be selective towards interferents such as alpha-fetoprotein, glucose, uric acid, bovine serum albumin and vitamin C [57].
Illustration of (a) 2D GR-Au and 3D GR-Au electrodes, (b) the formation of crumpled GR-Au composites via aerosol spray pyrolysis and (c) fabricating step of 3D label-free PSA immunosensor using crumpled GR-Au composites [
CA125 is a glycoprotein that exhibits abnormal levels in ovarian, breast, liver, lung and gastric cancer, serving as biomarker for the diagnosis and therapeutic monitoring of these diseases. Since its discovery in the ovarian epithelia by Bast
Efforts have been made to improve cost effectiveness and miniaturize diagnostic devices, as well as in multiplexing detection of several cancer biomarkers, which would reduce analysis costs and increase diagnose accuracy. Special attention has been given to microfluidic devices that reduce the volume of used reagents, are easy to use, transport and store, and give a fast analytical response. Paper-based platforms have also been investigated as low-cost and simple to use materials. For example, a 3D origami microfluidic paper-based immunosensor for simultaneous detection of CA125 and CEA using metal ions as electroactive labels was developed (Figure 7).
Origami EC immunodevice [
The support for the immobilization of captured Abs was constituted by a paper working electrode, modified with Au nanorods to ensure a large surface area and superior conductivity. For signal amplification, Au/BSA nanocarriers were used for loading Pd2+ and Cd2+, which were conjugated with anti-CA125 and anti-CEA for a sandwich assay. DPV measurements were recorded and the quantification of the two different Ags was obtained, based on the different stripping peaks obtained for the oxidation of the two metal tracers. LOD of 0.08 pg/mL and 0.06 mU/mL were obtained and the developed microfluidic POC device was successfully applied for the quantification of the biomarkers in human serum [58].
An interdigitated electrode-based capacitive sensor was reported for multiple biomarker detection: CEA, epidermal growth factor receptor (hEGFR) and CA15-3, based on AuNPs amplification. CEA and epidermal growth factor receptor (hEGFR) could be successfully detected in the concentration range of 20-1000 pg/mL, while cancer antigen 15-3 (CA15-3) was detected in the range of 10-200 U/mL, with LODs of 20 pg/mL and 10 U/ml, respectively [112].
Cardiovascular diseases include diseases of the heart and blood vessels and are a leading cause of death globally. The WHO estimated that deaths due to cardiovascular diseases in 2008 accounted for 30 % of worldwide deaths, and by 2030 the number of deaths per year will reach 23.3 million [113]. The main risk factors include smoking, alcohol abuse, hyperlipidaemia, high blood pressure, diabetes, physical inactivity and unhealthy diet. Myocardial infarct and stroke are acute events caused by obstructions of blood vessels or rupture of brain blood vessels. Rapid diagnosis in this case is crucial for the survival of patients. Beside signs and symptoms, current diagnosis of myocardial infarction is based on ECK and measurements of cardiac enzymes, while stroke is usually diagnosed using brain computed tomography, magnetic resonance imaging or carotid ultrasound. Other methods for diagnosis of cardiac conditions include echocardiography, chest X-ray, angiography or catheterization. Although ECK remains the main management method, some patients show normal ECK even in the presence of cardiopathies, and thus ECK may be inconclusive. Several markers exhibit elevated blood levels in the presence of heart conditions serving as diagnostic tools with important prognostic significance. As in the case of cancer, simultaneous multianalyte detection is preferred.
Myoglobin is released from injured muscle tissues and is a potential marker used in the diagnosis of myocardial infarction. However myoglobin is not specific for cardiac damage, and therefore it is usually used together with other biomarkers such as troponin or creatinine kinase MB subform (Ck-MB). Troponin I or T are proteins with important roles in muscle contraction that also detect cardiac damage as they are released in blood in elevated levels in case of cardiac muscle cell death. Ck-MB is an enzyme of the creatinine phosphokinase family specific to the heart cell; increased levels are present in case of heart muscle injuries. Serum levels of these markers rise rapidly within the first hours after a heart attack (myoglobin being the first to increase) reaching a maximum after several hours, depending on the size of the infarct [114]. Due to the fluctuation of the levels of cardiac markers, real-time monitoring is important for accurate measurements.
An SPR-based sensor for continuous monitoring of myoglobin levels in human serum was reported [59]. A recyclable Ab was immobilized at the surface of a label-free sensor that can quantify Ab-Ag reaction in real time. Two flow channels were used, one with specific Ab and the second, serving as control, with non-specific Ab. Buffered solutions and spiked serum samples of different concentration were then injected for the formation of immunocomplexes. The problems associated with the continuous sensing of myoglobin in human serum were overcome as follows: the non-specific binding of the analyte was avoided by pre-treating the sample with a mixture of detergents, specifically sodium dodecyl sulphate and P20, and adjusting the micelle size and net charge; and the aggregation of serum components was avoided by inactivating certain serum components by chelation of heavy metals. The sensor was able to detect the varying concentration of myoglobin over about 8 h with periodic one-point calibration every 3 h, with an LOD of 31.0 ng/mL, in the range from 31 ng/mL to 2000 ng/mL [58]. An electrochemical sensor for simultaneous detection of cardiac markers Troponin I (cTnI) and C-reactive protein (CRP), a known marker for inflammation and cardiac damage, has been reported based on a poly(dimethylsiloxane)-AuNP composite microfluidic system. Captured Abs for the two proteins were conjugated with CdTe and ZnSe QDs and square-wave anodic stripping voltammetry was used to detect the metal ions from the QDs in order to quantify the amount of bonded proteins. A linear range was obtained between 0.01 and 50 μg/L and 0.5 and 200 μg/L, with a very low LOD of approximately 0.004 μg/L and 0.22 μg/L, for cTnI and CRP, respectively [60]. Another target biomarker for cardiovascular disease is thrombin, a serine protease involved in blood coagulation, which acts as both a procoagulant (converting soluble fibrinogen in insoluble fibrin) and anticoagulant factor. It is also involved in the process of atherosclerosis and its cardiovascular complications [115]. The first Apt for thrombin, synthesized via SELEX in 1992, has a G-quadruplex structure and binds exosite I of thrombin [116]. A highly specific aptasensor was developed in a sandwich-type assay using planar Hall magnetoresistive transducer in combination with superparamagnetic labels. The sensor surface was modified with thiolated Apt to capture the thrombin added in the next step. Finally, a biotin-labelled secondary Apt was added to bind the thrombin-primary Apt complex and streptavidin-magnetic nanoparticles were used to measure the magnetoresistance signal. A linear response to thrombin concentration in the range of 86 pM-8.6 mM and an LOD as low as 86 pM was achieved using low sample volumes of only 2uL [61]. Interestingly, a simple Apt-target-Apt sensor was elaborated using the signal obtained after the electro-oxidation of intercalated MB and ferrocene for the quantification of thrombin. MB-tagged Apts were introduced by hybridization with complementary DNA sequences immobilized on the surface of the electrode. Thrombin was then added and its interaction with the Apt led to conformational changes of MB-Apt, driving the MB tag far from the electrode surface. A second Apt, tagged with ferrocene on both sides, was added to interact with thrombin, and the Apt’s conformational changes allow the two Fc tags to be close to the electrode surface. These conformational changes resulted in a decrease in the oxidation peak current of MB (
The detection of metabolites has gained increasing attention in recent years for the diagnosis of various diseases, metabolites being correlated with different pathologies. Glucose, for example, exhibits high levels in patients with diabetes and its monitoring plays a crucial role in the management of the disease. The devices used for the detection of glucose are mainly constituted by enzymatic sensors. The well-known glucometer accounts for 85 % of the market of commercially available biosensors [121]. Other metabolites of great interest are cholesterol and triglycerides, due to their involvement in atherosclerosis and in the pathology of cardiovascular diseases. Creatinine is a metabolite of creatine, eliminated by kidneys and excreted in urine, and it has become the preferred clinical marker for the diagnosis of renal filtration dysfunctions. The development of POC diagnostic devices could have an important impact on the improvement of the quality of life of patients suffering from metabolic disorders. Several POC devices for the detection of creatinine are already commercially available: IRMA TRUpoint (ITC, Edison, NJ), Radiometer ABL800 FLEX (Radiometer A/S, Bronshoj, Denmark), StatSensor (Nova Biomedical, Waltham, MA), and i-STAT (Abbott Diagnostics, East Windsor, NJ).
An interesting approach for detecting low-density lipoprotein (LDL) cholesterol was proposed based on immobilization of a large number of Abs on AuNP-silver chloride/polyaniline composite film. Abs specific to apolipoprotein 100 were chosen that recognized and bound apolipoprotein B present in the phospholipidic coats of the structure of LDL. EIS studies were performed to monitor the formation of Ab-Ag complex using [Fe(CN)6]4-/3- redox probe. An increase in the charge transfer resistance was observed after the binding of LDL, due to its low conductivity coupled with the repulsion between negatively-charged phospholipid units and the negative charge of the redox probe. This simple, label-free immunosensor allowed for the detection of low concentrations of LDL of 0.34 pg/mL, in the linear range of 0-33.5 pg/mL [63]. With a similar approach, an impedimetric label-free sensor was developed using aminated reduced graphene oxides for the immobilization of anti-apolipoprotein B on the electrode surface. The aminated graphene oxides were synthesized chemically and deposited on the surface of the transducers through electrophoretic deposition. EDC/NHS was then used for covalent attachment of Abs via amide bond formation, followed by the addition of LDL solutions. The change in the resistance after the formation of Ab-Ag complexes allowed for the detection of higher concentrations of LDL of 5mg/dL [64].
Besides metal nanoparticles and carbon-based nanomaterials, conductive polymers have been widely applied, for many years, as immobilization platforms for the development of immunosensors, due to their low cost, ease of preparation, and most importantly good biocompatibility. A simple electrochemical sensor was developed for the detection of creatinine in whole blood obtained from patients with renal transplant. The surface of a 16-gold electrode array was covered with a film of polypyrrole via electropolymerization, by applying a cyclic square-wave electrical field. Creatinine was embedded in the film in the same step. Anti-creatinine Abs labelled with HRP were mixed with a low volume of blood sample containing creatinine for the formation of immunocomplexes and added to the array for competitive reaction with the immobilized Ag. The decreased current of the HRP-Ab is proportional to the level of creatinine in the samples in the linear range of 0 mg/dL to 11.3 mg/dL with an LOD of 0.46 mg/dL. The method has the advantage of short response time <5 min and low sample volume (40 uL) [65].
Infectious diseases have increased rapidly in recent years, causing high fatality rates due to incorrect diagnosis, delay in treatment and various complications. Worldwide, infectious diseases account for nearly 40 % of the total 50 million annual estimated deaths, and represent the major cause of death in many developing countries [122].
Infections are caused by pathogenic microorganisms, such as bacteria, viruses, fungi or parasites. Most of the infections are highly contagious and can spread rapidly with the risk of causing epidemic or pandemic. Rapid and portable devices for the detection of pathogens are of paramount importance. Rapid screening for differentiation between viral and bacterial infection is extremely important for diagnosis and correct management of disease, limiting the overuse of antibiotics and reducing antibiotic resistance. A correct, rapid diagnosis may reduce the need for or period of hospitalization, with a high impact on medical costs. Current diagnosis is mostly based on cell culture and microscopic methods, serology or nucleic amplification testing, which are time-consuming. Most of the existing POC tests consist of immunoassays: agglutination, immunochromatographic and immunofiltration tests [123]. The development of portable devices that can be used at home or in doctors’ offices may permit a rapid diagnosis of various infections, allowing quick establishment of treatment and fast recovery by the patients. POC devices also have the advantages of being used in biological fluids with no need for complex sample pre-treatment.
Most of the sensors reported in the literature for the detection of pathogens are based on electrochemical techniques, due to their simplicity, low cost, rapid response and suitability for developing in small portable devices. The majority of these devices employ nucleic acids as recognition elements for the pathogenic agent, based on hybridization processes with a complementary DNA strand immobilized onto transducers, intercalation of redox mediators in double-stranded DNA, or label-free detection based mainly on impedimetric measurements.
Influenza is the most common acute respiratory infection, accounting for 52,294 deaths in the USA in 2011 [113]. It is caused by influenza type A, B and C viruses; the type A virus being responsible for the annually recurring epidemics. Mutations can also occur (e.g., avian or swine flu), raising health concerns.
A simple electrochemical DNA sensor was proposed based on avidin-biotin reaction for the immobilization of DNA capture probes on the surface of glassy carbon electrodes (GCEs). The surface of the electrode was firstly functionalized with carboxylic groups using 4-carboxyphenyl diazonium salts. Avidin was then bound, after ethylene carbodiimide/
Another label-free electrochemical immunosensor was reported for swine flu influenza virus (SIV), based on a self-assembled SWCNT network modified with anti-SIV Abs. The change in resistance upon binding of the Ag permits LODs of 180 TCID 50/mL (50 % Tissue Culture Infective Dose) to be achieved [124].
Piezoelectric techniques have attracted interest lately. A surface acoustic sensor based on Love waves was recently developed for the detection of influenza virus type A. SiO2coated piezoelectric LiNbO3 wafers were used to bind Ags against haemagglutinin (HA) of influenza A H1N1 subtype. After a silanization process of the wafers using tri-ethoxy-silyl-butyraldehyde and tri-ethoxy-silyl-undecanal ethylene-glycol-acetal, and the reaction of the silan groups with EDC/NHS, the substrates were immersed in a solution containing anti-HA Ags. Phycoerythrin-conjugated Abs were immobilized for fluorescence microscopy analysis in order to check the successful binding of HA. Non-conjugated Abs were used for surface acoustic wave testing giving an LOD of 1ng/mL [67].
Sexually-transmitted diseases (STDs) are viral or bacterial infections transmitted from one human to another through unprotected sexual contact. The most common viral STDs are HIV/AIDS, hepatitis B, herpes and human papillomavirus, while for bacterial infections the highest numbers of cases are diagnosed for gonorrhoea, chlamydia and syphilis. The incidence of these diseases is worryingly high; approximately 20 million people will contract an STD each year in the USA, and half of the infections occur in the age range 15-24 [125].
A simple device was developed for the detection of
Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS) and is transmitted mainly by unprotected sexual intercourse or use of unclean syringes [126].
A rapid, label-free capacitive immunosensor has been developed for real-time analysis of p24 Ag, a HIV-1 capsid protein that is detectable in serum shortly after infection. Silicon wafers were nanopatterned with gold via photolithography. A polytyramine film was then electrodeposited on the surface of the transducer, which chemisorbed AuNPs through the amine groups. Anti-HIV-1 p24 monoclonal Abs were then immobilized to capture the Ag from injected buffered or plasma samples, and the capacitance change was investigated. A linear relationship was observed from 2.4 10-6 to 2.4 10-3 pg/mL with an LOD of 7.9 10-8 pg/ml [70].
A simple electrochemical ELISA-type sensor for the detection of HIV-1 and HIV-2 Abs was recently reported. HIV-1 gp41 and HIV-2 gp36 Ags were immobilized at the surface of the transducers followed by the addition of anti-HIV Abs and IgG labelled with alkaline phosphatase. The substrate
Hepatitis is an inflammatory condition of the liver affecting many people worldwide, caused by various types of hepatitis viruses (A-E). Hepatitis B and C are chronic diseases causing cirrhosis and liver cancer [128].
An optical immunosensor for the detection of hepatitis C virus Abs was developed using the photoimmobilization method [129]. For this purpose, a photoactivatable electrogenerated polymer film was deposited on the surface of conductive fibre optics in order to link the bioreceptor to the fibre tip through light mediation, and a solution containing HCV-E2 envelope protein Ag was added and illuminated with UV light. Due to the photochemical reaction, a thin layer of the Ag is covalently attached to the polypyrrole-benzophenone-modified surface that is able to bind anti-E2 Abs from samples. Chemiluminescence measurements allowed anti-E2 Ig detection at a low titer [129].
A disposable impedimetric sensor was reported for the detection of
Therapeutic monitoring is important for drug adjustment to achieve optimum efficiency and minimal toxicity of the drug. Ideally, the concentrations of drugs should be measured at the site of the action - the receptor - but due to its inaccessibility, drug concentrations are measured in bodily fluids such as serum, plasma, saliva, urine or cerebrospinal fluids [131]. The most commonly used techniques for establishing drug levels for drug monitoring such as HPLC, GC-MS, LC-MA, radioimmunoassay, chemiluminescence, etc., are time- and reagent-consuming, require qualified personnel and complex sample pre-treatment, and are expensive. Therefore, there is an urge to develop new, minimally invasive devices for drug detection that could be easily used in homes, laboratories and clinics. Special attention should be given to targeting drugs with a narrow therapeutic range, which may be easily over- and under-dosed.
A simple ECL sensor was developed for the detection of clenbuterol, a drug used for pulmonary diseases, incorporating AuNPs and QDs for enhanced signals. The AuNP/ovalbumin-clenbuterol/anti-clenbuterol-QDs sensor was characterized using EIS and ECL emission achieving LOD of 0.0084 ng/mL in the range of 0.02-50 ng/mL [71]. An impedimetric sensor based on 3-mercaptopropionic acid SAMs was reported for the sensitive and selective detection of ketamine, an anaesthetic drug. The immobilization of Abs via SAM and the recognition of the target Ag were evaluated by the change in the charge transfer resistance, allowing for the detection of ketamine levels of 0.41 pmol/L [72]. EIS has also been employed for the detection of ciprofloxacin by a label-free immunosensor, which is widely used for the treatment of pulmonary, urinary or digestive infections. Polypyrrole-NHS electrogenerated films were used to immobilize anti-ciprofloxacin Abs, with an LOD of 10 pg/mL being obtained [73].
Developing new methods for drug quantification is also important in the quality control of drug formulations in the pharmaceutical industry. A label-free immunosensor based on the graphite oxide immobilization platform was proposed to quantify acetaminophen from pharmaceutical formulations. Anti-acetaminophen Abs were linked via amide groups by NHS/EDC chemistry on graphene oxide-modified SPEs. The preparation steps were investigated by several electrochemical techniques such as SWV, EIS and EQCM, and the LOD found by SWV measurements was 0.17 μM [74].
Another important social and health issue is
A simple and sensitive electrochemical sensor was developed for the simultaneous detection of two illicit drugs: morphine and methamphetamine. The simple approach employed SAM of 3-mercaptopropionic acid on a gold electrode that served to bind Abs specific for the targeted drugs. CV and EIS were employed as electrochemical techniques, revealing a decrease in the current together with an increase in the impedance after Ab binding and immunocomplex formation, due to the hindrance of electron transfer from the redox probe to the surface of electrodes. The sensors were able to detect morphine and methamphetamine in the linear range of 4 to 80 pg/L and 20-200 pg/L, respectively, with an LOD of 0.27 pg/L and 10.1 pg/L. The sensor is stable, reusable and may also be used in serum samples [133]. Another anti-doping device has been reported for the detection of human growth hormone, which is usually associated with anabolic steroids used to improve sporting performance. An SPR platform-based sandwich assay was developed using avidin-biotin coupling for the proper orientation of primary Abs, and the obtained sensor allowed the simultaneous detection of both hCG isoforms (20K and 22K) in concentrations as low as 0.9 ng/mL [134].
Detection of food contaminants is a major issue, since these contaminants may have serious consequences for food safety and human health, as well as for medical and economical costs. The rapid and sensitive detection of food contaminants is important to prevent intoxication and provide efficient treatment [135]. Diseases caused by food contaminants may vary widely from several million cases to 81 million cases in the USA, with bacterial foodborne outbreaks accounting for 91 % of the total outbreaks [122].
The most common foodborne pathogens include
An impedimetric immunosensor was developed for the detection of
An Apt-based biosensing approach based on an evanescent wave all-fibre (EWA) platform with embedded dethiobiotin-modified fibres was recently reported. In a simple target-capturing step using Apt-modified magnetic microbeads, signal probes (represented by Apt-complementary oligonucleotide) conjugated with streptavidin are released and further detected by an EWA biosensor via facial dethiobiotin streptavidin recognition. A reusable, sensitive sensor was thus obtained that can maintain its response after being reused 300 times, with an LOD of 3 nM in the linear range of 6 nM to 500 nM [75].
OTA was also detected in wine samples using two indirect competitive ELISA-type immunoassays coupled with electrochemical methods. pAb and mAb were investigated for the capture of the Ag and different enzymatic labels, horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were used and compared. The results showed that although similar performance was observed in buffer solution for the HRP-Ab-based strategy and the ALP-conjugated Ab sensor, when it comes to wine samples, the signal for the ALP-based sensor was affected by interferences. LODs of 0.7 ng/mL and 0.3 ng/mL were obtained for HRP- and ALP-labelled sensors [143].
Environmental air pollution and water control monitoring have become areas of intense interest in the research field due to the serious consequences for public health. Gas sensors are generally used for the detection of indoor and outdoor air pollutants such as carbon monoxide, formaldehyde, ethylene oxide, nitric oxide, hydrogen sulphide or chloride. Many contaminants infiltrate the soil, arriving in wastewaters and drinking water. These contaminants belong to various classes such as hormones, antibiotics, surfactants, endocrine disruptors, human and veterinary pharmaceuticals, X-ray contrast media, pesticides and metabolites or explosives, and are typically detected at concentrations ranging from 1 ng/L to 1 μg/L, reaching in some cases as much as 100 μg/L [144]. Water pollution is harmful to human, animal and water life, resulting in death of aquatic animals, disruption of food chains and diseases. The effects of consuming contaminating water can range from mild effects (diarrhoea, vomiting and nausea) to more severe diseases or even death. Pesticides, for example organochlorines, organophosphates, carbamates, pyretroides and atrazine, can cause neurological effects, asthma, allergies and endocrine disruption, and even cancer and reproduction problems. Other contaminants like nitroaromatic explosives, found in soil and groundwater near military zones, are toxic and carcinogenic to living organisms, causing aplastic anaemia, toxic hepatitis, hepatomegaly, cataract and skin irritation.
Standard methods for water quality analysis involve intensive sampling regimes and multistep sample preparation, requiring manual inputs which prohibit their integration into continuous monitoring systems, resulting in the need for developing new monitoring methods that can be easily integrated into water flow systems [145] such as electrochemical immunosensors.
Kanso’s group [76] reported on the development of electrochemical immunosensors based on magnetic beads (MBs) for the detection of oestradiol and ethinyl oestradiol in wastewaters. Oestradiol and ethinyl oestradiol are water contaminants found at levels of nanomolar range, which act as endocrine-disrupting chemicals, interfering with the endocrine systems and causing breast and testis cancers in humans. MBs were chemically modified with four different types of synthetic oestrogen derivative that contained carboxylic or amino groups. The assay was based on the competition between free oestrogen in the sample and immobilized oestrogen using a primary Ab labelled with ALP. Using SWV, an LOD of 1 and 10 ng/mL was obtained [76]. An interesting approach for a high-throughput microfluidic immunosensor for the detection of explosives in water has been presented by Adams et al. Immunoaffinity and fluorescence detection assays were coupled on a microfluidic device containing 39 parallel microchannels with immobilized Abs against TNT. With a total volume throughput of less than 6 mL/min, the assay is performed 60 times faster than the usual immunoassay, achieving an LOD of 0.01 ng/mL for TNT [77].
An electrochemical immunosensor for the simultaneous detection of pesticides, endosulphan and paraoxon was reported. Microcontact printing was used to pattern an SWCNT forest on the surface of transducers using diazonium salt chemistry. Redox probes of 1,1′-di(aminomethyl) ferrocene (FDMA) and pyrroloquinoline quinone (PQQ) were linked to the nanotubes, followed by Ab immobilization. Pesticides were quantified based on the current change of the redox probes upon the formation of immunocomplexes: the electrochemical signal of FDMA allowed the detection of endosulphan in the range of 0.05 to 100 ppb with an LOD of 0.05 ppb, while PQQ signal allowed paraoxon detection in the range from 2 to 2500 ppb with an LOD of 2 ppb [78].
New approaches using magnetic nanoparticles, quantum dots, carbon nanotubes, noble metal nanoparticles or hybrid nanomaterials, either as labels or immobilization platforms, have increased the number of electrochemical immunosensors and their applications. These materials offer important advantages, such as good biocompatibility, which maintains bioreceptor activity, a high surface-to-volume ratio, which increases the amount of immobilized bioreceptor, and unique conductivity and optical proprieties. Their high selectivity and sensitivity, as well as the possibility to miniaturize these systems, have made the use of electrochemical immunosensors for
Although numerous developments have been made in the field of immunosensors, novel techniques are still needed to improve the selectivity, sensitivity and simplicity of these devices, ready to meet the exigent requirements of clinical diagnosis or industry. The need for miniaturization and integration on an electronic platform able to detect the biomarkers or pathogens in real time are other challenges in this active area of research. In the past decade, remarkable progress in nanotechnology techniques has been made, together with new strategies using nanoparticles and nanostructured surfaces for electrochemical detection of a large variety of proteins. Low-cost, reliable, portable, multiplexed protein detection devices, especially those coupled with microfluidics, could ensure accurate detection of panels of cancer biomarkers in blood, urine or saliva. The application of immunosensors in drug abuse or doping as well as in the quantification of pathogens was achieved using various immobilization nanoplatforms for immobilization, and also using multienzyme labelling on metal nanoparticles, magnetic beads and carbon nanotubes or graphene, allowing for the ultrasensitive detection of target analytes.
The integration of electrochemical immunoassays into microfluidic platforms will create a versatile platform for the fabrication of devices for clinical diagnostics. The development and deployment of these systems could ultimately lead to more rapid clinical decision-making, reducing patient stress and health care costs.
In 1933 Fritz Zwicky [1] indicated a problem related to the galaxy cluster Coma. Galaxy cluster studied by Zwicky appeared to contain some 400 times more matter than an ordinary, visible, i.e., luminous matter. The content of the luminous matter was estimated form the amount of light emitted by the cluster. However, there was no response for that finding. Only 40 years later in 1970s the problem was rediscovered and concerned almost all of the galaxies. Research of Vera Rubin discovered that the galaxies rotate in a way that cannot be explained by taking into account visible, luminous matter. Today we know that most of the matter in the Universe is dark. Various attempts to resolve the problem of the existence of a mysterious form of matter, dark matter, have been taken ever since. One such idea is to find a particle to possibly complete the standard model. The most important property of such particle would be that it is not a subject to electromagnetic force; hence the dark matter is invisible in all electromagnetic wavelengths. In order to detect such particle, sensitive detectors are built, but still final conclusion has not been made. Another attempt of explaining the problem of missing matter was based on the assumption of existence of astrophysical objects such as black hole or dim brown dwarfs. This idea has rather been discredited as the abundance and masses of such objects are too small comparing to the amount of the matter that is missing. On different grounds stands the idea of modifying gravity in low acceleration regime. Modified Newtonian dynamics (MOND) proposed by M. Milgrom in 1983 is a phenomenological approach attempting to provide explanation of rotation of galaxies without invoking hidden matter at all. Yet such an approach seems to be in tension with recent findings of van Dokkum et al. about the ultra-diffuse galaxies. There appear to exist galaxies devoided of dark matter—then what about MOND predictions? This contribution is completed with the rotational curve of the Milky Way determined with 3 m in diameter radio telescope in the Astronomical Observatory of the Jagiellonian University. Obtained rotational curve is flat which indicates the presence of dark matter in the halo of our galaxy.
The term “dark matter” (DM) was introduced due to the contribution by Fritz Zwicky as early as in 1930s of the twentieth century. Studying the Coma cluster (of galaxies) located 320 million light-years away, Zwicky estimated [1] masses of the galaxies that make up this cluster based on the amount of light they emit. It turned out that such an amount of (
Figure schematically representing discrepancy between observed (B) and predicted (A) rotational curves of galaxies that indicates presence of dark matter in halos of such galaxies. Credit: PhilHibbs, Wikipedia,
Figure 1 illustrates this discrepancy. When being close to the center of the galaxy, the plot agrees with what one would expect: the rotational curve increases rapidly that reflects an obvious fact that the velocity of a test object (a “star”) increases as the effective gravitational force is growing (at a given radius, only the mass enclosed within a sphere of that radius is relevant in terms of excreting gravitational force—Newton’s Shell Theorem). Past a certain distance though (when increasing a distance from the massive center of galaxy does not enclose adequately bigger amounts of mass), the effective force of gravity should decline (as R2 will increase faster than the mass enclosed in a sphere of a radius being that distance from the center so the force of gravity will decline) which should result in lower orbital velocities.
Vera Rubin and Kent Ford published their first rotational curve in paper [2]. They presented there the rotation of Andromeda based on spectroscopic survey of emission regions applying neutral hydrogen, Hα, and [NII] λ6583 emission lines. Further works, see, e.g., [3], revealed that most of the galaxies have rather flat rotational curves like the one in Figure 1. The fact that more distant stars have almost constant velocity attracted the attention of scientists. The circular velocities of the stars are due to gravity which plays the role of centripetal force. Combining Newton’s law of gravity with an expression for centripetal force yields the following relation:
where
Since
As we know most of galaxies including the Milky Way have a bright massive center, a
Estimated distribution of matter and energy in the universe based on Planck data. Credit
Hypothetical particles that constitute the dark matter are called WIMPs which stands for weakly interacting massive particles. All the matter that we know (and us) is made of baryonic matter, i.e., the matter is made of baryons. WIMPS would be a new type of particles beyond the standard model. Those should be massive, subject to the gravitational force, and possibly other forces that are comparable to the weak force. One such candidate for WIMP could be a stable supersymmetric particle. Supersymmetric model has a particle of this property which was even called a “Wimp Miracle,” but we have not yet observed any trace of supersymmetry, moreover, Wimp Miracle in any of the particle colliders. WIMPs also should not interact via electromagnetism; hence the DM is not visible in any wavelength. We only can “see” the DM due to its gravitational interactions, which are strong enough to cause a phenomenon known as gravitational lensing.
This phenomenon is observed when the light rays passing near a very massive object are deflected (due to the curvature of space–time produced by this object) in such a way that a distant observer observes it lensed. Figure 3 illustrates gravitational lensing: the stretched structures are distant galaxies, whose light was bent by the DM between them and the observer. This allows to calculate the mass required to cause such phenomenon [6]. Large aggregations of massive DM particles are able to produce such image letting us to know it’s out there.
An image of gravitational lensing obtained with Hubble space telescope showing a distant image of galaxies which had been stretched due to the warping of space–time caused by a massive object between them and the observer.
Massive astrophysical compact halo objects (MACHOs) was another hypothesis invoked to explain the presence of large amount of nonluminous matter in galactic halos. Those, contrary to the WIMPS, would have been regular astrophysical objects emitting little or no radiation such as black holes, neutron stars, as well as brown dwarfs and unassociated planets, which drift unseen through interstellar space providing extra gravity. Thorough investigations have shown that this concept rather fails to explain the expected amount of the DM. One way to detect MACHOS’ influence, as described in [7], is to look for events of microlensing caused by them. Such microlensing would cause observable apparent amplification of star’s flux. In [7] it was shown that the number of such events is far too less that would have been expected. That rules out MACHOS as the candidates for DM. Moreover, the studies of abundance of baryons created in the Big Bang show that baryon density is consistent with the mean cosmic density of matter visible optically and in X-rays. It implies that most of the baryons in the Universe are visible but not dark and that most of the matter in the Universe consists of nonbaryonic DM [7].
In the former sections, we have discussed the attempts of solving or explaining the problem of the missing matter. That is to find or to claim existence of unknown, invisible substance. Yet there is another idea based on a different assumption. In 1983 Milgrom [8] proposed an idea that maybe it is the theory that needs to be modified rather than an invisible matter to be found. Modified Newtonian dynamics (MOND) is an empirically motivated modification of Newtonian dynamics at low accelerations, suggested as an alternative to dark matter concept [8, 9]. In Ref. [8] Milgrom considered the possibility that Newton’s second law does not describe the motion of objects under the conditions which prevail in galaxies and systems of galaxies. Newton’s laws have been tested in high-acceleration environment like the Earth or the solar system. The stars in the outer parts of the galaxies move in the circumstances of extremely low accelerations compared to what we know from everyday life. To illustrate how small such accelerations might be, let us calculate the acceleration of average star (the Sun) located on the suburbs of average galaxy (the Milky Way):
Milgrom proposed then a generalized form of Newton’s second principle, claiming the inertia term not to be simply proportional to the acceleration of an object but being rather a more general function of it:
In expression (5)
The acceleration constant is found to be
Recent studies of van Dokkum et al. [10, 11] have uncovered new class of object referred to as
Constraints on the intrinsic velocity dispersion of NGC1052-DF2. The result found in [
To give a reader some intuition and place this in some context, it is worth to notice that the stellar mass of the Milky Way found in [12] was
In 2012 Moni Bidin et al. [14] published a paper in which they estimated surface mass density in the solar neighborhood. Results obtained match the expectations of visible matter alone without the need of adding the dark matter component. The difference between the measured mass of matter (derived in this study) and the mass of visible matter (i.e., mass of matter that is estimated in the independent way based on the amount of emitted) provides an estimate of the amount of DM in the volume under analysis, and constraints on the shape of the DM halo can be derived. The fundamental basis for this measurement is the application of the Poisson–Boltzmann and Jeans equations to a virialized system in steady state. This allows to estimate either the local density at the solar position or the surface density (mass per unit area) of the mass within a given volume. Authors in Ref. [14] derive analytical expression for surface density as a function of distance from the galactic disk plane Σ(Z) to estimate the surface mass density between 1.5 and 4.5 kpc distance from the galactic disk plane using data from of the kinematics studies of about 400 red giants kinematics. The authors in [14] claimed that the estimate of the surface mass density matches the expectation of visible mass alone and the degree of overlap between the two curves is striking. There is no need for any dark component to account for the results: the measured Σ(Z) implies a local DM density
Observational results for the surface mass density, as a function of distance from the galactic plane (black curve), compared to the expectations of the models discussed in the text (thick gray curves). The dotted and dashed lines indicate the observational 1σ and 3σ strip, respectively. Expectations for the known visible mass are indicated by the thick gray curve labeled as VIS.
The experiments that aim at the direct detection due to scattering do not agree with each other yielding different constraints on the mass of the DM particles. The DAMA/LIBRA experiment [20] is the only one to claim positive result of detection which however has not been yet confirmed by the other groups (detectors). The aim of this experiment is detecting low-energy recoil photons from the scintillator crystals of thallium-doped sodium iodide NaI(Tl) placed in the detectors under the ground. Such photons would be emitted when the DM particle collides with one of the scintillators. If what we know about the DM is right, then since the Earth orbits the Sun, the DM particles should pass through the planet and hence have a chance to collide with those of the detectors. The idea of the experiment is that if one takes into account the revolution of the Earth around the Sun and the revolution of the Sun around the center of our galaxy, then the signals coming from the collisions should be modulated as in June the relative velocity of the Earth and the DM flux is the biggest hence yielding the biggest number of collisions. The data collected from the phase II of the experiment have all traits required to claim the presence of the DM in our part of the galaxy. The annual modulation is present only in the events concerning the photons with energies exactly within the energetic range theoretically predicted for the DM particles. Yet the DAMA/LIBRA is a singular case. Several groups have been working to develop experiments aiming at reproducing DAMA/LIBRA’s results using the same target medium. To determine whether there is evidence for an excess of events above the expected background in sodium iodide and to look for evidence of an annual modulation, the COSINE-100 experiment [21] uses the same target medium to carry out a model-independent test of DAMA/LIBRA’s claim. Their results from the initial operation of the COSINE-100 experiment were published in [21], and no excess of signal-like events above the expected background in the first 59.5 days of data from COSINE-100 has been observed. Assuming the so-called standard DM halo model, this result rules out spin-independent WIMP–nucleon interactions as the cause of the annual modulation observed by the DAMA/LIBRA collaboration. Another such experiment is the XENON100 experiment that searches for electronic recoil event rate modulation by measuring the scintillation light from a particle interacting in the liquid xenon. The results of this experiment published in [22] also exclude the DAMA/LIBRA results.
We will present here very briefly the other two methods of detection of DM:
DM manifests its existence through the shape of rotational curves of galaxies, in particular, through the rotational curve of our own galaxy, the Milky Way. This is what motivated us to take a glimpse on that topic and to compare results to those present in literature [23]. We have studied the rotational curve of Milky Way with radio telescope located in the Astronomical Observatory of the Jagiellonian University provided by EU-HOU project (EU-HOU project was founded with support from the European Commission, grant 510,308-LLP-1-2010-FR-COMENIUS-CMP. https://www.astro.uni-bonn.de/hisurvey/euhou/LABprofile/).
This 3 m in diameter telescope runs observations on 1420 MHz frequency which is the emission line of neutral hydrogen. When the hydrogen atom undergoes a transition from the state of higher energy when the spins of the proton and the electron are parallel to the state of lower energy that is when the spins are antiparallel, emitted photon is equivalent to radiation roughly 21 cm wavelength in vacuum (see Figure 6). Even though such process occurs very rarely, given the abundance of the hydrogen in the Universe (i.e., 74% of its baryonic mass), it is a common phenomenon. Hence the hydrogen is also present in the interstellar space around the stars, and radio observations yield information on how the matter is distributed inside the galaxy, and knowing the Doppler shift of the observed radiation, one can calculate the velocity of the hydrogen cloud from which it comes from. This in turn gives us an idea how the hydrogen and the nearby matter move within the galaxy, i.e., orbit around its center. Knowing the velocities and distance of such hydrogen clouds, one can plot the rotational curve of the galaxy. This is called tangent point method. Thus using the data obtained from the telescope, the Doppler equation:
Hydrogen 21-cm emission line.
one can calculate the source’s velocity (speed) relative to us (
To find the speed of the hydrogen cloud, a simple fact is used, that is the radial velocity results in difference between the projection of ours (Sun’s) velocity on the line of sight and the hydrogen cloud’s velocity on the line of sight (see Figure 7). The line of sight is determined along the galactic longitude (see Figure 8) on which we set the radio telescope.
Figure presenting two objects (A, B) along the line of sight. Hence object B lies in tangent point, i.e., its distance from the center of the galaxy is smaller, and its velocity is greater than the velocity of object A.
Figure presenting galactic longitude. L = 0° is direction from the solar system to the center of galaxy.
This results in the following equation for velocity of observed hydrogen cloud:
Among the objects observed along the given line of sight, the one with the smallest distance will have the biggest velocity. The smallest possible distance between us and the source is when it lies in the tangent point; hence simple trigonometry allows us to determine the distance:
which simplifies Eq. (8) to
Eqs. (8) and (9) provide all required information to plot a rotational curve of the galaxy. This method works for objects in I and IV Quadrants of galactic longitude, that is for
Twenty-nine objects with galactic longitude
Map of the hydrogen clouds used to determine the rotational curve of the Milky Way.
Rotational curve obtained from 21-cm line observations of the Milky Way. Note that the velocity of the studied objects appears to be constant over roughly 3 kpc distance.
Our rotational curve plot, Figure 10, is comparable to the plot obtained from data from LAB survey [24] and consistent with the ones that can be found in literature [23, 25]. We follow [25] in their choice of function to fit the data, namely
where we put
We conclude that the rotational curve reveals the existence of dark matter within the Milky Way. Taking (nonrelativistic) law of gravity, that is, the force of gravity is proportional to inverse squared distance, one would expect that the farther away the hydrogen clouds (constituting the distribution of matter) are from the massive center of the galaxy, the lower their velocities will be. As one see from the rotational curve, Figure 10, this is not the case; the velocities seem to be constant over a distance of roughly 3 kpc. Which means there is nonluminous matter distributed in such a way just to “keep up” with the increasing distance from the center of galaxy and make it so that the velocities of hydrogen atoms are almost constant as the distance increases.
The problem of missing matter discovered by Fritz Zwicky in 1933 appears to be still an open question. The most important premise of existence of the dark matter is the shape of rotational curves of galaxies, introduced as a tool for studying galaxy rotation by Vera Rubin. With our current understanding of the Universe, the dark matter, still a mysterious substance, makes up 86% of all the matter in the Universe. Throughout the years various attempts have been made to explain its nature. Some of the ideas have been proven unlikely (MACHOs). Some of them contradict each other (DAMA/LIBRA, the COSINE-100 collaboration). Yet even simple Milky Way’s observations as presented in Section 5 lead to the conclusion that the dark matter is present in the halo of our galaxy.
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Initial biochemical studies have been exclusively analytic: dissecting, purifying, and examining individual components of a biological system; in the apt words of Efraim Racker (1913 –1991), “Don’t waste clean thinking on dirty enzymes.” Today, however, biochemistry is becoming more agglomerative and comprehensive, setting out to integrate and describe entirely particular biological systems. The ‘big data’ metabolomics can define the complement of small molecules, e.g., in a soil or biofilm sample; proteomics can distinguish all the comprising proteins, e.g., serum; metagenomics can identify all the genes in a complex environment, e.g., the bovine rumen. 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Thus proteomics, an area of research that detects all protein forms expressed in an organism, including splice isoforms and post-translational modifications, is more suitable than genomics for a comprehensive understanding of the biochemical processes that govern life. The most common proteomics applications are currently in the clinical field for the identification, in a variety of biological matrices, of biomarkers for diagnosis and therapeutic intervention of disorders. From the comparison of proteomic profiles of control and disease or different physiological states, which may emerge, changes in protein expression can provide new insights into the roles played by some proteins in human pathologies. Understanding how proteins function and interact with each other is another goal of proteomics that makes this approach even more intriguing. Specialized technology and expertise are required to assess the proteome of any biological sample. Currently, proteomics relies mainly on mass spectrometry (MS) combined with electrophoretic (1 or 2-DE-MS) and/or chromatographic techniques (LC-MS/MS). MS is an excellent tool that has gained popularity in proteomics because of its ability to gather a complex body of information such as cataloging protein expression, identifying protein modification sites, and defining protein interactions. 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