ELISA is seen as the state-of-the-art technique for highly sensitive serological diagnosis. It is based on the interaction of a pathogenic antigen and its corresponding antibodies.
The typically used “sandwich” ELISA requires an antigen with at least two binding sites and a pair of antibodies binding these sites. At first, a capture antibody is immobilized on the surface of a microwell plate.
After incubation with the sample and binding of the respective antigen, the secondary antibody is added. If the antigen was present in the sample, the secondary antibody or a third one, binding the second, yields a signal enhancement. Detection usually happens via enzyme coupled reactions. This procedure increases the sensitivity about 10,000 fold down to pg/ml scales
12)
.
ELISA is a very sensitive and specific test, most commonly used in serological diagnostics, e.g. for Varicella Zoster
13)
,
Hepatitis B
14)
,
Toxoplasmosis
15)
or Ebola
16)
.
Depending on the assay protocol used, a whole ELISA can be carried out within some hours to one day.
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{{Freiburg/Menubar}} | {{Freiburg/Menubar}} | ||
− | {{Freiburg/ | + | {{Team:Freiburg/wiki_content_start_bubble}} |
<html> | <html> | ||
<style> | <style> | ||
+ | |||
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/*========= BEGIN: style for navigation bar ==========*/ | /*========= BEGIN: style for navigation bar ==========*/ | ||
#project { | #project { | ||
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/*========= END: style for navigation bar ==========*/ | /*========= END: style for navigation bar ==========*/ | ||
+ | horizontal_menu { | ||
+ | margin: 0px auto; | ||
+ | padding: 5%; | ||
+ | } | ||
+ | |||
+ | .horizontal_menu ul{ | ||
+ | margin: 0px auto; | ||
+ | padding: 0px; | ||
+ | } | ||
+ | |||
+ | .horizontal_menu li { | ||
+ | margin: 5px auto; | ||
+ | padding: 2.1%; | ||
+ | width: 120px; | ||
+ | list-style: none; | ||
+ | display: inline-block; | ||
+ | position: relative; | ||
+ | text-align: center; | ||
+ | border: 2px groove #0051A2; | ||
+ | border-radius: 8px; | ||
+ | } | ||
/*h2{ | /*h2{ | ||
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</style> | </style> | ||
− | < | + | <script type="text/javascript"> |
+ | //===================BEGIN:Amazing Bubble Sidebar========================== | ||
− | + | $(document).ready(function(){ | |
− | + | // CHANGE THE FOLLOWING ATTRIBUTES // | |
− | + | var href_text1='https://2015.igem.org/Team:Freiburg/Project/Overview', | |
− | + | // Text2 needs no href as it is the actual page // | |
− | + | img_url='https://static.igem.org/mediawiki/2015/7/76/Freiburg_icon_project_white_03.png', | |
+ | href_text3='https://2015.igem.org/Team:Freiburg/Project/DNA_Engineering', | ||
+ | // Text1 needs no text as it is a pic // | ||
+ | text2='Diagnostics Today'; | ||
+ | // Text3 needs no text as its always 'next' // | ||
+ | // HOLD ON CHANGING THINGS --JABBERWOCK // | ||
− | + | $('#bubble1').attr('href',href_text1); | |
+ | $('#bubble1_img').attr('src', img_url); | ||
+ | $('#bubble3').attr('href',href_text3); | ||
− | + | $('#bubble2').text(text2); | |
− | + | }); | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | //===================END:Amazing Bubble Sidebar========================== | |
− | + | </script> | |
− | + | ||
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− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | </ | + | |
+ | <div class="content_box"> | ||
+ | <h1>Today's Applications of Immunodiagnostics</h1> | ||
− | |||
− | <div class=" | + | <div class="horizontal_menu"> |
− | + | <ul> | |
+ | <li><a href="#Introduction">Introduction<br> </a></li> | ||
+ | <li><a href="#Current_Methods">Current Methods and Limitations<a></li> | ||
+ | <li><a href="#Limitations">Requirements<br> </a></li> | ||
+ | <li><a href="#DiaCHIP_Contribution">DiaCHIP Contribution</a></li> | ||
+ | <li><a href="#Outlook">Outlook<br> </a></li> | ||
+ | </ul> | ||
</div> | </div> | ||
− | + | </div> | |
− | + | ||
− | + | ||
+ | <div class="content_box"> | ||
+ | <!-- EDIT3 SECTION "What are current diagnostic methods" [3045-11352] --> | ||
+ | <span id="Limitations" class="anchor"></span> | ||
+ | <h2>Requirements for Future Diagnostic Devices</h2> | ||
+ | |||
+ | <div class="image_box left"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/7/7a/Freiburg_sanduhr.jpg" width="150px"> | ||
+ | </div> | ||
+ | |||
+ | <p> | ||
+ | Nowadays, a lot of diseases can be treated well. A crucial factor fast diagnosis essential for an immediate onset of appropriate treatment. | ||
+ | It depicts a critical factor for the patient's health and life and significantly determines his well-being. | ||
+ | Moreover, improved diagnostics are not only required regarding the health of a patient: 70% of healthcare expenses are linked to diagnostic tests | ||
+ | <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a> | ||
+ | </sup>. | ||
+ | Therefore, improvements in diagnostic technologies have the potential to drastically reduce overall healthcare costs while at the same time increasing health as such. | ||
+ | </br> | ||
+ | </br> | ||
+ | Diagnostic tests are usually developed for use in air-conditioned laboratories with refrigerated storage of chemicals, a constant supply of calibrators and reagents, highly trained personal and rapid transportation of samples. | ||
+ | This setting is not available for most developing countries | ||
+ | <sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a> | ||
+ | </sup>. | ||
+ | Thus, most of the substantial progress achieved in the public health and Point-of-Care sector has only been advantageous to the more developed part of the world. | ||
+ | </br> | ||
+ | </p> | ||
+ | |||
<div class="image_box right"> | <div class="image_box right"> | ||
− | + | <div class="thumb2 tcenter" style="width:370px"> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <div class=" | + | <div class="thumbinner"><a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/8/81/Freiburg_DEATHne.png" title="https://static.igem.org/mediawiki/2015/8/81/Freiburg_DEATHne.png"> |
− | + | <img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/8/81/Freiburg_DEATHne.png" width="350"/></a> | |
− | + | <div class="thumbcaption"> | |
− | + | <div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/8/81/Freiburg_DEATHne.png" title="vergrößern"> | |
− | + | <img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a> | |
− | + | </div> | |
− | + | </div> | |
− | + | </div> | |
− | + | </div> | |
− | + | ||
</div> | </div> | ||
− | + | <p> | |
− | <p> | + | According to the WHO 2.5 out of 6 billion people lack basic sanitation, 2 billions do not have access to electricity and more than 1 billion lack basic healthcare services and clean drinking water |
− | According to the WHO <sup><a class="fn_top" href="# | + | <sup><a class="fn_top" href="#fn__3" id="fnt__3" name="fnt__3">3)</a> |
− | Moreover, 50% of all deaths in the most impoverished developing countries are a result of infectious diseases, whereas in the wealthiest developed countries this concerns less than 5% | + | </sup>. |
− | </ | + | Moreover, 50% of all deaths in the most impoverished developing countries are a result of infectious diseases, whereas in the wealthiest developed countries this concerns less than 5% |
− | </br> | + | <sup><a class="fn_top" href="#fn__4" id="fnt__4" name="fnt__4">4)</a></sup>. |
− | Therefore, transforming existing technologies into | + | <br> |
− | </p> | + | Therefore, transforming existing technologies into portable applications is a leap forward to improve general health all over the world. These applications should be robust and sensitive enough for the use outside of equipped laboratories. Outbreaks and spreading of potential epidemic diseases or sexually transmitted infections could be controlled by a rapid diagnosis and immediate onset of appropriate treatment |
+ | <sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a></sup>. | ||
+ | </p> | ||
− | <div class="image_box left"> | + | <div class="image_box left"> |
− | + | <div class="thumb2 tcenter" style="width:250px"> | |
− | </div> | + | <div class="thumbinner"><a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/d/db/Freiburg_STDs.png" title="https://static.igem.org/mediawiki/2015/d/db/Freiburg_STDs.png"> |
+ | <img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/d/db/Freiburg_STDs.png" width="250"/></a> | ||
+ | <div class="thumbcaption"> | ||
+ | <div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/d/db/Freiburg_STDs.png" title="vergrößern"> | ||
+ | <img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <p> | ||
+ | A need for such technologies is urgent: 500 million people between the age of 15 and 49 are infected with curable sexually transmitted infections like chlamydia, gonorrhea, <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Diseases#Syphilis" title="syphilis">syphilis</a> or trichomoniasis each year | ||
+ | <sup><a class="fn_top" href="#fn__5" id="fnt__5" name="fnt__5">5)</a> | ||
+ | </sup>! | ||
+ | However, the infrastructure currently available for the diagnosis of infectious diseases often proves to be too slow and expensive to be practicable for third world countries. | ||
+ | This can be illustrated with the identification of pathogens of an infectious diarrhea taking 2-4 days – even in the best developed laboratories of the world | ||
+ | <sup><a class="fn_top" href="#fn__6" id="fnt__6" name="fnt__6">6)</a> | ||
+ | </sup>. | ||
+ | </p> | ||
+ | |||
+ | <p> | ||
+ | According to the problems and needs discussed, devices for future diagnostics should meet the following requirements: | ||
− | < | + | <ul> |
− | + | <li> <strong>Speed</strong> - a fast diagnosis reduces time until the beginning of treatment, preventing the spread of epidemic diseases and reducing the severity of a disease | |
+ | </li> | ||
+ | </br> | ||
+ | <li> <strong>Simplicity</strong> - the handling necessary to perform the test should be as easy as possible | ||
+ | </li> | ||
+ | </br> | ||
+ | <li> <strong>Low-cost</strong> - Point-of-Care diagnostics need to be affordable in developing countries | ||
+ | </li> | ||
+ | </br> | ||
+ | <li > <strong>Unambiguity</strong> - the output of Point-of-Care tests requires the clarity of a yes/no answer | ||
+ | </li> | ||
+ | </br> | ||
+ | <li> <strong>Storage under sub-optimal conditions</strong> - since defined conditions may not be available, the device has to be stable under extreme conditions concerning temperature, pH or humidity for example | ||
+ | </li> | ||
+ | </br> | ||
+ | <li> <strong>Multiplexing</strong> - covering a broad spectrum of possible diseases in only one device allows for a differential diagnosis with the need for only one sample of the patient | ||
+ | </li> | ||
+ | </br> | ||
+ | </ul> | ||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | <div class="content_box"> | ||
+ | <!-- EDIT1 SECTION "Today's Applications of Immunodiagnostics" [1-206] --> | ||
+ | <span id="Introduction" class="anchor"></span> | ||
+ | <h2 class="sectionedit2">The Field of Diagnostics</h2> | ||
+ | |||
+ | <p> | ||
+ | Medical diagnostics comprise the whole process of tracing the source leading to a patient's symptoms. Usually, it is understood as the identification of a pathogen or a malfunction responsible for the illness. <br> | ||
+ | To achieve an efficient disease treatment, clinical diagnostics are mostly divided into the following steps: | ||
+ | </p> | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>After interviewing the patient and considering his medical history, risk factors and current problems, a clinician proposes a certain differential diagnosis - thus pre-limiting the spectrum of possible diseases.<br><br> | ||
+ | </li> | ||
+ | <li>This is mostly followed by the performance of diagnostic tests (usually in a central laboratory) to confirm the differential diagnosis and to clearly identify - or at least further limit - possible causes of the symptoms.<br><br> | ||
+ | </li> | ||
+ | <li >Finally, this leads to a treatment consisting of medication, surgery, hospitalization or discharge.<br> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | <div class="image_box right"> | ||
<br> | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/3/3a/Freiburg_Patient.jpeg" width="350px"> | ||
+ | </div> | ||
+ | <p> | ||
+ | Interest in the so called Point-Of-Care (POC) diagnostics increased dramatically | ||
+ | <sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a> | ||
+ | </sup> | ||
+ | <sup><a class="fn_top" href="#fn__7" id="fnt__7" name="fnt__7">7)</a> | ||
+ | </sup> | ||
+ | in the last decades. POC diagnostics (or bedside diagnostics) are diagnostic tests that can be performed directly at a patient's site or even bedside. The output of such a test is immediately available, circumventing the usually necessary sending of samples to external labs. | ||
+ | The term POC encompasses many possible end-use settings outside of a centralized testing facility like emergency settings, regional health clinics, medical practices as well as home or mobile use. <br> | ||
+ | These tests are devices that are present in everyday life, such as AB0-testing, blood glucose testing, blood gas and electrolytes analysis, pregnancy testing and cholesterol screening. <br> | ||
+ | For the near future an increase in the amount of products for POC diagnostic is predicted | ||
+ | <sup><a class="fn_top" href="#fn__8" id="fnt__8" name="fnt__8">8)</a> | ||
+ | </sup>, | ||
+ | thereby confirming the need for such applications. | ||
+ | In the face of aging populations, spreading of infectious diseases especially in the developing world, biohazard threats and increasing numbers of autoimmune diseases and allergies in the developed world | ||
+ | <sup><a class="fn_top" href="#fn__9" id="fnt__9" name="fnt__9">9)</a> | ||
+ | </sup> | ||
+ | <sup><a class="fn_top" href="#fn__10" id="fnt__10" name="fnt__10">10)</a> | ||
+ | </sup>, | ||
+ | POC tests become inevitable. | ||
+ | </p> | ||
+ | </div> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | < | + | <div class="content_box"> |
− | < | + | <!-- EDIT2 SECTION "What are diagnostics" [207-3044] --> |
+ | <span id="Current_Methods" class="anchor"></span> | ||
+ | <h2>Current Diagnostic Methods and Limitations</h2> | ||
+ | <p> | ||
+ | The four most common centralized laboratory techniques are blood chemistry, immunoassays, nucleic-acid amplification tests and flow cytometry | ||
+ | <sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a> | ||
+ | </sup>. | ||
+ | As the | ||
+ | <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/System" title="DiaCHIP">DiaCHIP | ||
+ | </a> | ||
+ | is an immunodiagnostic method, we will focus on this part of diagnostics and compare it to commonly applied methods in today's clinics and labs. Immunodiagnostics are based on antigen-antibody interactions, which might be present within the body fluids of a patient. By detecting and identifying key proteins within a patient's sample like blood or urine, these tests enable to distinguish between major classes of diseases like infectious, metabolic, or cardiovascular diseases or cancer | ||
+ | <sup><a class="fn_top" href="#fn__7" id="fnt__7" name="fnt__7">7)</a> | ||
+ | </sup>. | ||
+ | Immunodiagnostics are realized by immunoassays, which summarize a wide range of formats, allowing quantification and monitoring of small molecules, large proteins and even whole pathogens | ||
+ | <sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a>. | ||
+ | </sup> | ||
+ | Three prominent examples of immunoassays are lateral flow tests, ELISAs and miniaturized immunoassays (microarrays). | ||
+ | <br> | ||
+ | </p> | ||
− | < | + | <div class="image_box right"> |
− | </ | + | <img src="https://static.igem.org/mediawiki/2015/b/b7/Freiburg_Diagnostics_ELISA.jpg" width="120px"> |
+ | </div> | ||
− | <p> | + | <p> |
− | + | <strong>ELISA - sensitive but time consuming, no multiplexing</strong>: | |
− | </ | + | </br> |
+ | <br> | ||
+ | The commonly used ELISA (enzyme linked immunosorbent assay) only provides a limited capacity for multiplexing as only one specific disease can per detected per well. It takes several hours and large amounts of sample material as well as antibodies (0.05-1.2 µg antibody per well | ||
+ | <sup><a class="fn_top" href="#fnt__11" id="fn__11" name="fn__11">11)</a> | ||
+ | </sup> | ||
+ | ) to test for all the possible diseases fitting the patient's symptoms. | ||
+ | <div class="accordion"> | ||
+ | <div class="accordion-section"> | ||
+ | <a class="accordion-section-title" href="#accordion-1">More information</a> | ||
+ | <div id="accordion-1" class="accordion-section-content" style="display:none; padding:15px;"> | ||
+ | <p> | ||
+ | </br> | ||
+ | ELISA is seen as the state-of-the-art technique for highly sensitive serological diagnosis. It is based on the interaction of a pathogenic antigen and its corresponding antibodies.</br> | ||
+ | The typically used “sandwich” ELISA requires an antigen with at least two binding sites and a pair of antibodies binding these sites. At first, a capture antibody is immobilized on the surface of a microwell plate. <br> | ||
+ | After incubation with the sample and binding of the respective antigen, the secondary antibody is added. If the antigen was present in the sample, the secondary antibody or a third one, binding the second, yields a signal enhancement. Detection usually happens via enzyme coupled reactions. This procedure increases the sensitivity about 10,000 fold down to pg/ml scales | ||
+ | <sup><a class="fn_top" href="#fn__12" id="fnt__12" name="fnt__12">12)</a> | ||
+ | </sup>. | ||
+ | ELISA is a very sensitive and specific test, most commonly used in serological diagnostics, e.g. for <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Diseases#Zoster">Varicella Zoster</a> | ||
+ | <sup><a class="fn_top" href="#fn__13" id="fnt__13" name="fnt__13">13)</a> | ||
+ | </sup>, | ||
+ | Hepatitis B | ||
+ | <sup><a class="fn_top" href="#fn__14" id="fnt__14" name="fnt__14">14)</a> | ||
+ | </sup>, | ||
+ | Toxoplasmosis | ||
+ | <sup><a class="fn_top" href="#fn__15" id="fnt__15" name="fnt__15">15)</a> | ||
+ | </sup> | ||
+ | or Ebola | ||
+ | <sup><a class="fn_top" href="#fn__16" id="fnt__16" name="fnt__16">16)</a> | ||
+ | </sup>. | ||
+ | Depending on the assay protocol used, a whole ELISA can be carried out within some hours to one day. | ||
+ | <br> | ||
+ | </p> | ||
+ | </div><!-- end accordion-section-content --> | ||
+ | </div><!-- end accordion-section --> | ||
+ | </div> <!-- end accordion --> | ||
+ | </p> | ||
− | + | <div class="image_box left"> | |
+ | <img src="https://static.igem.org/mediawiki/2015/6/62/Freiburg_Schwangerschaftstest.jpeg" width="120px"> | ||
+ | </div> | ||
+ | |||
+ | <p> | ||
+ | <strong>Lateral flow test - simple and rapid, no multiplexing, limited sensitivity</strong> | ||
+ | <br> | ||
+ | <br> | ||
+ | Lateral flow tests are much faster to perform but can only detect one molecule of interest at once. Moreover, they are known to perform rather poorly in terms of sensitivity. | ||
− | <div class=" | + | <div class="accordion"> |
− | < | + | <div class="accordion-section"> |
+ | <a class="accordion-section-title" href="#accordion-2">More information</a> | ||
+ | <div id="accordion-2" class="accordion-section-content" style="display:none; padding:15px;"> | ||
+ | <p> | ||
+ | <br> | ||
+ | The lateral flow test is commonly produced as strip test. It is rather complicated in its setup, but extremely easy to use as only one drop of liquid has to be added to get a result that is easy to interpret within minutes. The test confirms its validity and the presence or absence of the target molecule through the appearance of colored stripes. Prominent examples are pregnancy tests, drug-abuse tests and HIV diagnostics in developing countries | ||
+ | <sup><a class="fn_top" href="#fn__8" id="fnt__8" name="fnt__8">8)</a> | ||
+ | </sup> | ||
+ | or blood-glucose tests. Stripe tests can be seen as the gold standard for Point-of-Care devices - easy to store, use and easy to read out. | ||
+ | </br> | ||
+ | </p> | ||
+ | </div><!-- end accordion-section-content --> | ||
+ | </div><!-- end accordion-section --> | ||
+ | </div> <!-- end accordion --> | ||
− | <div class=" | + | </p> |
− | <a | + | |
− | + | <p> | |
+ | <strong>Miniaturized Immunoassay (Microarray) and Lab-on-a-Chip (LOC)</strong> | ||
+ | <br> | ||
+ | <br> | ||
+ | Miniaturized immunoassays (microarrays) combined with microfluidic bioanalysis have been shown to hold great potential regarding future diagnostics | ||
+ | <sup><a class="fn_top" href="#fn__7" id="fnt__7" name="fnt__7">7)</a> | ||
+ | </sup>. | ||
+ | In comparison to ELISAs and lateral flow tests, they already hold the possibility for multiplexing. | ||
+ | |||
+ | |||
+ | <div class="accordion"> | ||
+ | <div class="accordion-section"> | ||
+ | <a class="accordion-section-title" href="#accordion-3">More information</a> | ||
+ | <div id="accordion-3" class="accordion-section-content" style="display:none; padding:15px;"> | ||
+ | <p> | ||
+ | Lab-on-a-Chip (LOC) refers to the idea that many processes in the lab can be improved and automated by miniaturizing them in one chip. A microarray is a multiplexed or multi-parallel LOC-device | ||
+ | <sup><a class="fn_top" href="#fn__17" id="fnt__17" name="fnt__17">17)</a> | ||
+ | </sup>. | ||
+ | <br> | ||
+ | Many scientists consider LOC-based methods to be the most promising technological advance to fundamentally transform the Point-Of-Care diagnostic industry | ||
+ | <sup><a class="fn_top" href="#fn__8" id="fnt__8" name="fnt__8">8)</a> | ||
+ | </sup> | ||
+ | <sup><a class="fn_top" href="#fn__18" id="fnt__18" name="fnt__18">18)</a> | ||
+ | </sup>. | ||
+ | Nowadays, miniaturized immunoassays are one of the most important analysis platforms for proteins | ||
+ | <sup><a class="fn_top" href="#fn__7" id="fnt__7" name="fnt__7">7)</a> | ||
+ | </sup>. | ||
+ | <br> | ||
+ | The development of Lab-on-a-Chip systems is closely linked to the emergence of microfluidics. Microfluidic techniques use small, compact, low-power and mass-producible chips, which are designed for small sample sizes and rapid but at the same time sensitive analysis | ||
+ | <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a> | ||
+ | </sup>. | ||
+ | Various LOC diagnostic modules have been integrated within microfluidic chips, providing devices with immense multiplexing probabilities and high functionality | ||
+ | <sup><a class="fn_top" href="#fn__8" id="fnt__8" name="fnt__8">8)</a> | ||
+ | </sup>. | ||
+ | </br> | ||
+ | However, up to date complex microfluidics and LOC systems have not yet fulfilled people’s expectations to revolutionize the healthcare industry. Nevertheless, more simplistic lateral flow assays are a huge success. | ||
+ | </br> | ||
+ | For diagnostic systems, a broad and diverse field of methods and techniques is available. As this is far more than we are able to describe here, we want to refer to the overview article of Roth <i>et al.</i> | ||
+ | <sup><a class="fn_top" href="#fn__19" id="fnt__19" name="fnt__19">19)</a> | ||
+ | </sup>. | ||
+ | </p> | ||
+ | </div><!-- end accordion-section-content --> | ||
+ | </div><!-- end accordion-section --> | ||
+ | </div> <!-- end accordion --> | ||
+ | </p> | ||
+ | |||
+ | <p> | ||
+ | <strong>Addressable Laser Bead Immunoassays</strong> | ||
+ | <br> <br> | ||
+ | Laser and microsphere based immunoassays obtain a possibility of multiplexing. | ||
+ | Since they are based on the immobilization of antigens on microbeads and subsequent analysis using laser technology, they require high-tech equipment and trained personnel. | ||
+ | |||
+ | <div class="accordion"> | ||
+ | <div class="accordion-section"> | ||
+ | <a class="accordion-section-title" href="#accordion-4">More information</a> | ||
+ | <div id="accordion-4" class="accordion-section-content" style="display:none; padding:15px;"> | ||
+ | <p> | ||
+ | In this diagnostic device different antigens are immobilized on microbeads. | ||
+ | Those exhibit laser-reactive colors, each related to a distinct antigen and thus maintaining the identity information | ||
+ | <sup><a class="fn_top" href="#fn__20" id="fnt__20" name="fnt__20">20)</a> | ||
+ | </sup>. | ||
+ | The microspheres are mixed after coating and incubated with a serum sample and a fluorescence labeled secondary antibody. | ||
+ | The analysis is conducted by performing dual laser flow cytometry. | ||
+ | A first laser gathers the identity information of the microbead's color and the second the fluorescence intensity. | ||
+ | </p> | ||
+ | </div><!-- end accordion-section-content --> | ||
+ | </div><!-- end accordion-section --> | ||
+ | </div> <!-- end accordion --> | ||
+ | |||
+ | |||
+ | </p> | ||
+ | |||
+ | <br> | ||
+ | <br> | ||
+ | <p> | ||
+ | Even though current diagnostic methods provide reliable information on a broad range of diseases, they still encounter various restrictions. | ||
+ | As already mentioned, time and the possibility of multiplexing are some crucial issues regarding future diagnostics. | ||
+ | For instance, ELISAs do not provide a possibility for multiplexing and performing the test lasts at least some hours. | ||
+ | <br> | ||
+ | Especially for the last mentioned methods, a lot of expensive equipment is needed for analyzing the results. | ||
+ | This leads either to conflicts regarding involved costs or the size of most devices that are mostly far from portable. | ||
+ | This restricts the use to developed countries and special facilities. | ||
+ | <br> | ||
+ | Besides those complications, all the methods described above rely on a solid material with immobilized proteins on it. | ||
+ | Purified proteins are generally known to be difficult to handle as they are rather unstable. | ||
+ | The storage of proteins therefore requires particular conditions that are not easy to provide in some parts of the world. | ||
+ | </p> | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | <div class=" | + | <div class="content_box"> |
− | <a | + | <span id="DiaCHIP_Contribution" class="anchor"></span> |
− | + | <h2 class="sectionedit5">How Can Our DiaCHIP Contribute to Solving These Problems?</h2> | |
− | </ | + | <p> |
+ | Our approach, the <a href="https://2015.igem.org/Team:Freiburg/Project/System">DiaCHIP</a>, basically combines <strong>three promising techniques</strong> in <strong>one device</strong>, offering great potential to improve future diagnostics. | ||
+ | </p> | ||
+ | |||
+ | <a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics" title="multiplexing">Miniaturized Immunoassays Combined with Microfluidics</a> | ||
+ | <p> | ||
+ | Miniaturized immunoassays enable immense multiplexing. By immobilizing hundreds of different antigens it is possible to screen a patient's sample for the existence of many potential antibodies. This allows customized combinations of antigens, thereby providing the optimal detection system for the respective application.<br> | ||
+ | For the microfluidic device only small volumes of reagents and samples are necessary making it cost-efficient additionally to saving time. | ||
+ | </p> | ||
+ | |||
+ | <a href="https://2015.igem.org/Team:Freiburg/Project/Cellfree_Expression" title="cell_free">Microarray Copying (Generating Protein Arrays From DNA Array Templates)</a> | ||
+ | <p> | ||
+ | Storage and handling problems of conventional peptide based microarrays are circumvented by directly producing our protein array on the basis of a DNA array template via cell-free expression. | ||
+ | As DNA is stable within a large range of temperatures, pH values, and other environmental conditions it proves to be an ideal molecule for storing protein information. | ||
+ | </p> | ||
+ | |||
+ | <a href="https://2015.igem.org/Team:Freiburg/Project/iRIf" title="iRif">iRIf Detection Method</a> | ||
+ | <p> | ||
+ | This emerging detection method enables a fast, sensitive and label-free detection of binding processes. | ||
+ | In our setup, antibodies present in the patient's blood bind to the antigens produced via cell-free expression and can be detected in real-time. | ||
+ | Incubation steps with secondary detection antibodies (that have to be labelled in some way) are for example rendered unnecessary, thus making detection cheaper and faster. | ||
+ | Nevertheless, if the signal should be intensified, flushing the array with a secondary antibody can be performed. | ||
+ | This would extend the time needed for testing by a maximum of half an hour. | ||
+ | </p> | ||
+ | |||
+ | <a href="https://2015.igem.org/Team:Freiburg/Results/Own_Device" title="iRif">Small Detection Device</a> | ||
+ | <p> | ||
+ | To enable many people to benefit from this promising detection method we rebuilt this device ourselves. | ||
+ | In contrast to the commercial device it is now even smaller than a shoebox and built of simple components. | ||
+ | </p> | ||
− | |||
− | |||
</div> | </div> | ||
− | <div class=" | + | <div class="content_box"> |
− | <a class=" | + | <span id="Outlook" class="anchor"></span> |
− | + | <h2 class="sectionedit6">Outlook</h2> | |
− | + | <p> | |
− | <a | + | The future of diagnostics may lie in home-care devices based on microfluidic Lab-on-a-Chip systems. |
− | + | These are supposed to perform assays at a sensitivity, specificity and reproducibility similar to those of central laboratory analysis. | |
+ | However, the user should only need to apply one drop of blood. | ||
+ | <br> | ||
+ | Especially people in developing countries could perform routine testing to detect the infection with pathogens like influenza or sexually transmittable diseases like <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Diseases#AIDS" title="AIDS">AIDS</a> or <a clas="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Diseases#Syphilis" title="Syphilis">syphilis</a> | ||
+ | <sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a> | ||
+ | </sup>. | ||
+ | <br> | ||
+ | The DiaCHIP may be scaled down to a size suitable for smaller medical practices or portable applications. | ||
+ | Thereby, it complements the existing techniques that are either small and handy but only able to detect a limited spectrum of antibodies or are so huge that an efficient use is only possible in clinical facilities. | ||
+ | We showed the basic feasibility of such a device with our <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Results/Own_Device" title="own_device">rebuilt setup</a> and the successful <a href="https://2015.igem.org/Team:Freiburg/Results">detection of anti-tetanus antibodies in human blood serum</a>. | ||
+ | <br> | ||
+ | Core characteristics of the self-built device are its simplicity, the low cost of the components and the rapid generation of an evaluable result. | ||
+ | Even though the DiaCHIP is still at an experimental stage, some improvements may render it easy to handle even for untrained users. | ||
+ | </p> | ||
+ | <br> | ||
+ | <br> | ||
+ | On this page, we only focused on diagnostics as this is the main application we suppose. Nevertheless, the methodology behind the DiaCHIP can be used for <a href="https://2015.igem.org/Team:Freiburg/Project/Future_Directions">further applications</a>. | ||
</div> | </div> | ||
+ | <div class="content_box"> | ||
+ | <div class="footnotes"> | ||
+ | <h3>References</h3> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__1" id="fn__1" name="fn__1">1)</a></sup> | ||
+ | <a target="_Blank" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3991778/"> | ||
+ | Mao & Huang, 2012. Exploiting mechanical biomarkers in microfluidics. Lab Chip.</a> | ||
+ | </div> | ||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__2" id="fn__2" name="fn__2">2)</a></sup> | ||
+ | <a target="_Blank" href="http://www.nature.com/nature/journal/v442/n7101/abs/nature05064.html"> | ||
+ | Yager et al., 2006. Microfluidic diagnostic technologies for gloal public health. Nature.</a> | ||
+ | </div> | ||
− | </ | + | <div class="fn"> |
+ | <sup><a class="fn_bot" href="#fnt__3" id="fn__3" name="fn__3">3)</a></sup> | ||
+ | <a target="_blank" href="http://apps.who.int/iris/bitstream/10665/81965/1/9789241564588_eng.pdf"> | ||
+ | WHO, 2013. World Health Statistics Report.</a> | ||
+ | </div> | ||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__4" id="fn__4" name="fn__4">4)</a></sup> | ||
+ | <a target="_Blank" href="http://www.who.int/whr/2002/en/whr02_en.pdf?ua=1"> | ||
+ | WHO, 2002. World Health Report.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__5" id="fn__5" name="fn__5">5)</a></sup> | ||
+ | <a target="_blank" href="http://apps.who.int/iris/bitstream/10665/75181/1/9789241503839_eng.pdf"> | ||
+ | WHO, 2008. Global incidence and prevalence of selected curable sexually transmitted infections.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__6" id="fn__6" name="fn__6">6)</a></sup> | ||
+ | <a class="urlextern" href="http://cid.oxfordjournals.org/content/32/3/331.short"> | ||
+ | Guerrant et al., 2001. Practice guidelines for the management of infectious diarrhea. Clinical Infectious Diseases.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__7" id="fn__7" name="fn__7">7)</a></sup> | ||
+ | <a target="_Blank" href="http://onlinelibrary.wiley.com/doi/10.1002/adma.201100464/abstract"> | ||
+ | Gervais et al., 2011. Microfluidic Chips for Point-of-Care Immunodiagnostics. Adv. Mater.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__8" id="fn__8" name="fn__8">8)</a></sup> | ||
+ | <a target="_Blank" href="http://pubs.rsc.org/en/content/articlehtml/2012/lc/c2lc21204h"> | ||
+ | Chin et al., 2011. Commercialization of microfluidic point-of-care diagnostic devices. Royal Society of Chemistry.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__9" id="fn__9" name="fn__9">9)</a></sup> | ||
+ | <a target="_Blank" href="http://www.ncbi.nlm.nih.gov/pubmed/?term=9130472"> | ||
+ | Ring, 1997. Allergy and modern society: Does “‘Western life style’” promote the development of allergies? Int. Arch. Allergy Immunol.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__10" id="fn__10" name="fn__10">10)</a></sup> | ||
+ | <a target="_Blank" href="http://onlinelibrary.wiley.com/doi/10.1046/j.1365-3083.1999.00640.x/abstract"> | ||
+ | Arbuckle et al., 1999. The development of lupus humoral autoimmunity for anti-Sm autoantibodies is consistent with predictable sequential B-cell epitope spreading. Scand J Immunol.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__11" id="fn__11" name="fn__11">11)</a></sup> | ||
+ | <a target="_Blank" href="http://tools.thermofisher.com/content/sfs/brochures/TR0065-ELISA-guide.pdf"> | ||
+ | Thermo Fischer Scientific Inc., 2010. ELISA technical guide and protocols.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__12" id="fn__12" name="fn__12">12)</a></sup> | ||
+ | <a target="_Blank" href="http://www.clinchem.org/content/22/8/1243.abstract"> | ||
+ | Wisdom, 1976. Enzyme-immunoassay. Clinical Chemistry.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__13" id="fn__13" name="fn__13">13)</a></sup> | ||
+ | <a target="_Blank" href="http://link.springer.com/article/10.1007%2Fs00430-006-0032-z"> | ||
+ | Sauerbrei et al., 2006. Herpes simplex and varicella-zoster virus infections during pregnancy: current concepts of prevention, diagnosis and therapy. Part 2: Varicella-zoster virus infections. Med Microbiol Immunol.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__14" id="fn__14" name="fn__14">14)</a></sup> | ||
+ | <a target="_Blank" href="http://www.sciencedirect.com/science/article/pii/S0166093499000397"> | ||
+ | Usuda et al., 1999. Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product. Journal Virol. Methods.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__15" id="fn__15" name="fn__15">15)</a></sup> | ||
+ | <a target="_Blank" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395899/"> | ||
+ | Carlier et al., 1980. Evaluation of the enzyme-linked immunosorbent assay (ELISA) and other serological tests for the diagnosis of toxoplasmosis. Bull. World Health Organization.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__16" id="fn__16" name="fn__16">16)</a></sup> | ||
+ | <a target="_Blank" href="http://jid.oxfordjournals.org/content/179/Supplement_1/S192.long"> | ||
+ | Ksiazek et al., 1999. ELISA for the detection of antibodies to Ebola viruses. Journal of Infectious Diseases</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__17" id="fn__17" name="fn__17">17)</a></sup> | ||
+ | <a target="_Blank" href="http://www.epa.gov/radiation/docs/cleanup/nanotechnology/chapter-3-lab-on-a-chip.pdf"> | ||
+ | Lab-on-a-Chip systems.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__18" id="fn__18" name="fn__18">18)</a></sup> | ||
+ | <a target="_Blank" href="http://www.biochemia-medica.com/content/ilza-salamunic-laboratory-diagnosis-autoimmune-diseases-new-technologies-old-dilemmas"> | ||
+ | Salamunic, 2009. Laboratory diagnosis of autoimmune diseases – new technologies, old dilemmas. Biochemia Medica.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__19" id="fn__19" name="fn__19">19)</a></sup> | ||
+ | <a target="_Blank" href="http://pubs.rsc.org/en/content/articlepdf/2010/cs/b820557b"> | ||
+ | Roth et al., 2009. Microfluidic lab-on-a-chip platforms: requirements, characteristics and applications. Chemical Society Reviews.</a> | ||
+ | </div> | ||
+ | |||
+ | <div class="fn"> | ||
+ | <sup><a class="fn_bot" href="#fnt__20" id="fn__20" name="fn__20">20)</a></sup> | ||
+ | <a target="_blank" href="http://www.pabst-publishers.de/Medizin/buecher/3899671481_i.htm"> | ||
+ | Fritzler, 2014. New technologies in the detection of autoantibodies: Evaluation of addressable laser bead immunoassays (ALBIA).</a> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | <!-- Labjournal content ends here --> | ||
{{Freiburg/wiki_content_end}} | {{Freiburg/wiki_content_end}} |
Latest revision as of 03:21, 19 September 2015
![](https://static.igem.org/mediawiki/2015/8/85/Freiburg_wiki_background_2_parted_left.png)
![](https://static.igem.org/mediawiki/2015/f/f4/Freiburg_wiki_background_2_parted_right.png)
Today's Applications of Immunodiagnostics
Requirements for Future Diagnostic Devices
![](https://static.igem.org/mediawiki/2015/7/7a/Freiburg_sanduhr.jpg)
Nowadays, a lot of diseases can be treated well. A crucial factor fast diagnosis essential for an immediate onset of appropriate treatment. It depicts a critical factor for the patient's health and life and significantly determines his well-being. Moreover, improved diagnostics are not only required regarding the health of a patient: 70% of healthcare expenses are linked to diagnostic tests 1) . Therefore, improvements in diagnostic technologies have the potential to drastically reduce overall healthcare costs while at the same time increasing health as such. Diagnostic tests are usually developed for use in air-conditioned laboratories with refrigerated storage of chemicals, a constant supply of calibrators and reagents, highly trained personal and rapid transportation of samples. This setting is not available for most developing countries 2) . Thus, most of the substantial progress achieved in the public health and Point-of-Care sector has only been advantageous to the more developed part of the world.
According to the WHO 2.5 out of 6 billion people lack basic sanitation, 2 billions do not have access to electricity and more than 1 billion lack basic healthcare services and clean drinking water
3)
.
Moreover, 50% of all deaths in the most impoverished developing countries are a result of infectious diseases, whereas in the wealthiest developed countries this concerns less than 5%
4).
Therefore, transforming existing technologies into portable applications is a leap forward to improve general health all over the world. These applications should be robust and sensitive enough for the use outside of equipped laboratories. Outbreaks and spreading of potential epidemic diseases or sexually transmitted infections could be controlled by a rapid diagnosis and immediate onset of appropriate treatment
2).
A need for such technologies is urgent: 500 million people between the age of 15 and 49 are infected with curable sexually transmitted infections like chlamydia, gonorrhea, syphilis or trichomoniasis each year 5) ! However, the infrastructure currently available for the diagnosis of infectious diseases often proves to be too slow and expensive to be practicable for third world countries. This can be illustrated with the identification of pathogens of an infectious diarrhea taking 2-4 days – even in the best developed laboratories of the world 6) .
According to the problems and needs discussed, devices for future diagnostics should meet the following requirements:
- Speed - a fast diagnosis reduces time until the beginning of treatment, preventing the spread of epidemic diseases and reducing the severity of a disease
- Simplicity - the handling necessary to perform the test should be as easy as possible
- Low-cost - Point-of-Care diagnostics need to be affordable in developing countries
- Unambiguity - the output of Point-of-Care tests requires the clarity of a yes/no answer
- Storage under sub-optimal conditions - since defined conditions may not be available, the device has to be stable under extreme conditions concerning temperature, pH or humidity for example
- Multiplexing - covering a broad spectrum of possible diseases in only one device allows for a differential diagnosis with the need for only one sample of the patient
The Field of Diagnostics
Medical diagnostics comprise the whole process of tracing the source leading to a patient's symptoms. Usually, it is understood as the identification of a pathogen or a malfunction responsible for the illness.
To achieve an efficient disease treatment, clinical diagnostics are mostly divided into the following steps:
- After interviewing the patient and considering his medical history, risk factors and current problems, a clinician proposes a certain differential diagnosis - thus pre-limiting the spectrum of possible diseases.
- This is mostly followed by the performance of diagnostic tests (usually in a central laboratory) to confirm the differential diagnosis and to clearly identify - or at least further limit - possible causes of the symptoms.
- Finally, this leads to a treatment consisting of medication, surgery, hospitalization or discharge.
![](https://static.igem.org/mediawiki/2015/3/3a/Freiburg_Patient.jpeg)
Interest in the so called Point-Of-Care (POC) diagnostics increased dramatically
2)
7)
in the last decades. POC diagnostics (or bedside diagnostics) are diagnostic tests that can be performed directly at a patient's site or even bedside. The output of such a test is immediately available, circumventing the usually necessary sending of samples to external labs.
The term POC encompasses many possible end-use settings outside of a centralized testing facility like emergency settings, regional health clinics, medical practices as well as home or mobile use.
These tests are devices that are present in everyday life, such as AB0-testing, blood glucose testing, blood gas and electrolytes analysis, pregnancy testing and cholesterol screening.
For the near future an increase in the amount of products for POC diagnostic is predicted
8)
,
thereby confirming the need for such applications.
In the face of aging populations, spreading of infectious diseases especially in the developing world, biohazard threats and increasing numbers of autoimmune diseases and allergies in the developed world
9)
10)
,
POC tests become inevitable.
Current Diagnostic Methods and Limitations
The four most common centralized laboratory techniques are blood chemistry, immunoassays, nucleic-acid amplification tests and flow cytometry
2)
.
As the
DiaCHIP
is an immunodiagnostic method, we will focus on this part of diagnostics and compare it to commonly applied methods in today's clinics and labs. Immunodiagnostics are based on antigen-antibody interactions, which might be present within the body fluids of a patient. By detecting and identifying key proteins within a patient's sample like blood or urine, these tests enable to distinguish between major classes of diseases like infectious, metabolic, or cardiovascular diseases or cancer
7)
.
Immunodiagnostics are realized by immunoassays, which summarize a wide range of formats, allowing quantification and monitoring of small molecules, large proteins and even whole pathogens
2).
Three prominent examples of immunoassays are lateral flow tests, ELISAs and miniaturized immunoassays (microarrays).
![](https://static.igem.org/mediawiki/2015/b/b7/Freiburg_Diagnostics_ELISA.jpg)
ELISA - sensitive but time consuming, no multiplexing:
The commonly used ELISA (enzyme linked immunosorbent assay) only provides a limited capacity for multiplexing as only one specific disease can per detected per well. It takes several hours and large amounts of sample material as well as antibodies (0.05-1.2 µg antibody per well
11)
) to test for all the possible diseases fitting the patient's symptoms.
![](https://static.igem.org/mediawiki/2015/6/62/Freiburg_Schwangerschaftstest.jpeg)
Lateral flow test - simple and rapid, no multiplexing, limited sensitivity
Lateral flow tests are much faster to perform but can only detect one molecule of interest at once. Moreover, they are known to perform rather poorly in terms of sensitivity.
Miniaturized Immunoassay (Microarray) and Lab-on-a-Chip (LOC)
Miniaturized immunoassays (microarrays) combined with microfluidic bioanalysis have been shown to hold great potential regarding future diagnostics
7)
.
In comparison to ELISAs and lateral flow tests, they already hold the possibility for multiplexing.
Addressable Laser Bead Immunoassays
Laser and microsphere based immunoassays obtain a possibility of multiplexing.
Since they are based on the immobilization of antigens on microbeads and subsequent analysis using laser technology, they require high-tech equipment and trained personnel.
Even though current diagnostic methods provide reliable information on a broad range of diseases, they still encounter various restrictions.
As already mentioned, time and the possibility of multiplexing are some crucial issues regarding future diagnostics.
For instance, ELISAs do not provide a possibility for multiplexing and performing the test lasts at least some hours.
Especially for the last mentioned methods, a lot of expensive equipment is needed for analyzing the results.
This leads either to conflicts regarding involved costs or the size of most devices that are mostly far from portable.
This restricts the use to developed countries and special facilities.
Besides those complications, all the methods described above rely on a solid material with immobilized proteins on it.
Purified proteins are generally known to be difficult to handle as they are rather unstable.
The storage of proteins therefore requires particular conditions that are not easy to provide in some parts of the world.
How Can Our DiaCHIP Contribute to Solving These Problems?
Our approach, the DiaCHIP, basically combines three promising techniques in one device, offering great potential to improve future diagnostics.
Miniaturized Immunoassays Combined with Microfluidics
Miniaturized immunoassays enable immense multiplexing. By immobilizing hundreds of different antigens it is possible to screen a patient's sample for the existence of many potential antibodies. This allows customized combinations of antigens, thereby providing the optimal detection system for the respective application.
For the microfluidic device only small volumes of reagents and samples are necessary making it cost-efficient additionally to saving time.
Storage and handling problems of conventional peptide based microarrays are circumvented by directly producing our protein array on the basis of a DNA array template via cell-free expression. As DNA is stable within a large range of temperatures, pH values, and other environmental conditions it proves to be an ideal molecule for storing protein information.
iRIf Detection MethodThis emerging detection method enables a fast, sensitive and label-free detection of binding processes. In our setup, antibodies present in the patient's blood bind to the antigens produced via cell-free expression and can be detected in real-time. Incubation steps with secondary detection antibodies (that have to be labelled in some way) are for example rendered unnecessary, thus making detection cheaper and faster. Nevertheless, if the signal should be intensified, flushing the array with a secondary antibody can be performed. This would extend the time needed for testing by a maximum of half an hour.
Small Detection DeviceTo enable many people to benefit from this promising detection method we rebuilt this device ourselves. In contrast to the commercial device it is now even smaller than a shoebox and built of simple components.
Outlook
The future of diagnostics may lie in home-care devices based on microfluidic Lab-on-a-Chip systems.
These are supposed to perform assays at a sensitivity, specificity and reproducibility similar to those of central laboratory analysis.
However, the user should only need to apply one drop of blood.
Especially people in developing countries could perform routine testing to detect the infection with pathogens like influenza or sexually transmittable diseases like AIDS or syphilis
2)
.
The DiaCHIP may be scaled down to a size suitable for smaller medical practices or portable applications.
Thereby, it complements the existing techniques that are either small and handy but only able to detect a limited spectrum of antibodies or are so huge that an efficient use is only possible in clinical facilities.
We showed the basic feasibility of such a device with our rebuilt setup and the successful detection of anti-tetanus antibodies in human blood serum.
Core characteristics of the self-built device are its simplicity, the low cost of the components and the rapid generation of an evaluable result.
Even though the DiaCHIP is still at an experimental stage, some improvements may render it easy to handle even for untrained users.
On this page, we only focused on diagnostics as this is the main application we suppose. Nevertheless, the methodology behind the DiaCHIP can be used for further applications.