By allowing us insight into ever smaller and more complex systems, nanotechnology is revolutionizing our world. Nanotechnology provides novel tools in understanding the chemical and molecular building blocks of the human body. From medicines to diagnostics, the scope of nanotechnological applications in biomedicine has evolved in leaps and bounds unveiling novel methodologies to improve quality of life. Engineered particles allow for site-specific drug delivery while Superparamagnetic Iron Oxides (SPIO) particles tagged to cells allow us to utilize MRI to track cell migration in vivo (Baumjohann et al, 2006). While nanotechnology allows medicine to become more personalized, in our ever more global society, it is essential to utilize nanotechnology for the improvement of public health. Indeed, such dynamic and proactive research will maximally impact both individual and population health driving nanomedicine into the new century (Brenner, 2010). Small portable diagnostic technologies will be one of the most enabling systems in culturing a healthier global population.
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Early diagnosis is often the best indicator in disease progression (Diamandis, 2002). While the past century has seen amazing leaps in diagnostic technologies leading to longer lifespans and improved quality of life, these diagnostic technologies are often only available in developed countries. The inequality of medical access between sociopolitical climes is a threat to overall global health. Not only does limited medical technology harm the populations lacking the technology, but when individuals migrate to developed countries they may bring with them undiagnosed infectious disease or chronic conditions, which may burden the national healthcare system. Improved access to medicine begins with improved and earlier diagnosis; however, many developing countries lack the facilities, skills, and finances in which to perform complex diagnostic testing (Helmann, 2007).
Nano diagnostic Lab- on 'a-Chip (LOC) systems integrates micro and nanotechnologies with biological systems to perform portable laboratory functions (Kalyaniwalla, 2005). Such systems hold promise in bettering world health by providing real time diagnostic testing where such testing is difficult to perform. Fast complex testing is useful for clinicians in -field to allocate drugs and track disease progress in resource-restricted areas. Early diagnosis allows for the best prognosis and often the cheapest course of treatment, factors important in public health measures.
Nationally, LOC systems offer to provide early detection of chronic diseases such as cancer. Pancreatic cancer is a highly fatal carcinoma with a mortality rate at five years of approximately 96% (Von Hoff et all, 2005). By exploiting the properties of nanoscale diagnostics, small quantities of antigens can be detected in the body which is essential in detecting and characterizing early stage cancers. Early detection through primary care screenings in cancers ultimately lowers healthcare costs associated with tertiary care procedures. Improving access to primary care to prevent and treat disease rapidly is a major goal in public health.
There has been much progress made in the field of nanotechnology over the past two decades due to the availability of enabling technologies. The field has the potential to quickly and sensitively detect tumor producing antigens earlier than ever improving prognosis of previously fatal diseases. Such state of the art screening and prevention methodologies will drive both the fields of public health and nanomedicine in decades to come.
Pancreatic cancer is an extremely devastating form of cancer with a 5-year survival rate of roughly 5%. Its death rate virtually equals its incidence rate (Broudo, 2010). The term pancreatic cancer is rather vague because there are several types of Pancreatic Cancer; the most common and most deadly type is exocrine pancreatic cancer (which is the target of the biosensor). The main issue with this disease is that early detection of pancreatic cancer is very difficult to accomplish. According to Surveillance, Epidemiology, and End Results (SEER) those found in the first stage (IA) have a 10-year survival rate of 30.6% while those diagnosed later in stages such as IIA or III have a 10-year survival rate of 8.6% and 1.4% respectively (Key, 2002). One ideal solution to creating a higher survival rate through early detection is to use a biosensor to sense the biomarkers presented by a developing pancreatic tumor.
A biomarker by definition is a substance or process that suggests the presence of cancer, which can be either a molecule produced by the tumor or a response by the body's defense system that indicates the presence of a tumor (Verma, 2010). Several biomarkers that are present in the early development of pancreatic cancer are carbohydrate antigen 19-9 (CA 19-9), carcinoembryonic antigen (CEA) and cell adhesion molecule 17.1 (CAM 17.1). CA 19-9 is paired with rabbit monoclonal 1116-NS-19-9 (IgG1) (19-9 Ab) antibody. This antibody is related to the Lewis-A blood group antigen and showed to be 90% specific and 90% sensitive (Moghimi, 2007). However, approximately 7% of the population is Lewis negative, which has the potential to create a false negative; this promotes the use of another biomarker on the biosensor. The second biomarker carcinoembryonic antigen (CEA) is paired with mouse monoclonal CEA antibody [CI-P83-1] (IgG1) (ABCAM). which has 84% sensitivity and 90% specificity (Moghimi, 2007). When CEA and Ca 19-9 are used together, 100% specificity was revealed. This increase in specificity is because CEA is able to distinguish between the Lewis negative populations (Moghimi, 2007). The final biomarker is aimed at increasing the overall sensitivity and selectivity of the biosensor for assurance of detection. Rat monoclonal antimucin CAM 17-1 is an antimucin antibody, which normally binds mucin. CAM 17-1 is found in the pancreas and senses the presence of sialyl I blood-group antigen, which is present in the case of a tumor (Yiannakou, 1997). CAM 17-1 offers 91% specificity and 86% sensitivity, the advantage of using CAM 17-1 with CEA and Ca 19-9 is that it would add another measure of specificity. Additionally, the use of CAM 17-1 will increase sensitivity due to the fact that it can detect Lewis negative individuals. (Hoff, Evans, and Hruban, 2005; Diamandis et al, 2002)
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The biosensor in question will be a nano diagnostic Lab-on-a-Chip (LOC) system, which tests for tumor biomarkers. The structure and function of the LOC system involves a chamber where human blood sample will flow through micro and nanofluidic channels into a chamber, after which antibody probes will subsequently be injected. The first step involves a human sample that is injected into the channel, which is filtered; parts of human sample are absorbed on the surface. The remaining surface is blocked to minimize nonspecific absorption of the target antibody (Pereira, 2011). The next step in the LOC system is the injection of probe antibodies, which will bind to the antigens attached to the surface. The concentration of antigens bound to the surface is vital for the reaction (Pereira, 2011). An electric field is applied within the chamber to aid in movement of injected fluids. Secondary antibodies are then allowed to react with the primary antibodies bound to the immobilized antigen probes. These secondary antibodies are fluorescently marked using QD to aid in detection. All three antigens will be associated with three different QD that fluoresce at different wavelengths of light. The LOC system can then be analyzed using an optical filter, only allowing a certain wavelength of light through to the chip which will allow only the specific QD complex to fluoresce.
A LOC system biosensor offers many advantages. Not only does it bring hope to the fight of pancreatic cancer but it also shows promise for other difficult to detect diseases. This is true even in the third world, where expensive testing prevents individuals from being tested for the various diseases that they are at risk for. The LOC system offers a solution due to the disposable nature of several components of the LOC system biosensor. If the biosensor LOC system is positive, the patient will immediately be sent to have a multiphasic helical CT scan and then an endoscopic ultrasound with a fine needle aspiration (EUS+FNA) ; this is done to determine if there is indeed a tumor and if a resection can be done (Santo, 2004). The benefit of the biosensor is that it is less invasive and will only call for EUS+FNA and a CT scan if necessary. This is a much more practical and cost effective approach for proper early detection of pancreatic cancer.
Cancer by definition is a disease that if not caught early will be exponentially more devastating to the individual affected; this cannot be any more accurate than in the case of pancreatic cancer. Individuals that are at high risk for pancreatic cancer, such as those with a family history of pancreatic cancer, smokers, those who suffer from chronic pancreatitis, and those with mucinous cysts should be tested on a regular basis. The LOC biosensor offers an advantage in the fight against pancreatic cancer; its multiple antibody setup creates a high specificity and sensitivity. Thusly, this biosensor, when applied to individuals that have high risk factors and are tested routinely, has the capacity to either save lives or increase quality of life in said individuals. Overall, a 10-year 30.6% survival rate at stage IA or even 19.2% at stage IB as opposed to 0.3% at stage IV justifies the production and continued use of this biosensor (Key, 2002).
Fabrication and Design:
A lab on a chip immunoassay involving micro and nanofluidics for testing of serological tumor markers was proposed for early detection of pancreatic cancer. Specificity, sensitivity, portability, speed, and cost were aspects considered in product development. This bioanalytical technology filters fluid, immobilizes the antigen, permits an antibody antigen reaction, and fluoresces in the event of positive antibody recognition.
Polydimethylsiloxane (PDMS) is an inert, nontoxic polymeric elastomeric silicon. With a potential resolution down to 6nm through soft lithography, it is the ideal material to create micro to nano sized fluid channels. Micro and nanochannels were formed using soft lithography in PDMS off of a silicon base substrate coated in SU 8 photoresist. After applying a shadow mask UV is used to pattern the SU 8 photoresist on top of its silicon base. The resulting microchannels were approximately 20'm wide by 200'm long. After formation of the molds, PDMS was injected on top of the silicon mold and cured. After removal from the mold, the PDMS base was approximately 50'm thick. Nanoscale channels are then etched on top of the soft lithographed microchannels using electron beam lithography on a thin film resist to pattern pores under 20nm in diameter and 200'm length. The patterned PDMS layer is integrated onto a glass chip by treating both the glass and PDMS inside a plasma cleaner for 30 seconds. Pressing the two materials together post treatment should irreversibly bind them together forming the complete lab on a chip measuring 10mm width by 35mm length (Walker, 2003;. One input and one output for the fluids measuring 2mm in diameter each are situated on top of the lab on a chip.
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The final step in creation of this nanofluidic immunoassay is the visualization of test results to indicate a positive reaction. Quantum dot (QD) complexes bound to secondary antigens will emit light upon antigen-antibody conjugation. A 200'm by 200'm photodiode was created out of hydrogenated amorphous silicon (Si:H) and fabricated on a glass substrate to detect QD emissions. On top of the glass, an aluminum electrode is placed and patterned using wet-etching. A mesa-type PIN semiconductor system was deposited on silane through plasma assisted vapor deposition through the deposition of p region diborane and n region phosphine. The PN junction was physically limited by creating a mesa through photolithography. Next, a 100nm insulating layer of silicon nitride. Finally, a 2'm silicon carbon amorphous alloy was used as a filter layer for the QD fluorescence. Approximately 20 of these photodiodes were placed on the chip 200'm apart (Pereira et al, 2011). The excitation light source was coupled to a monochromator to select the incident wavelengths of 625nm, 705nm, and 565nm.
Nanofluidics allow for superior performance in lab on a chip devices compared to microfluid conduits in sample preparation, fluid handling and injection, separation, and detection. While serological samples can be prepared off chip, in-chip track etched membranes (TEMs) were layered between fluid channels inside the chip to prevent blocked channels and encourage even flow (Ferain et al, 1997; Kovarik et al ,2009). A polycarbonate TEM with homogenous 15nm diameter nanopore distributions was added in between two of the channels to separate out undesired proteins and other biologics. At the interface where microchannels filter out into nanopores an applied electric field generated from thin film gold electrodes concentrated the analyte at the anode end of the channel. Anions become concentrated at the cathode due to electrostatic exclusion from an unbalanced cationic flux. As cations move toward the cathode, anions form an ion depletion region which concentrates the analyte at the anode (Kovarik et al, 2009). This concentration of the analyte aids in test sensitivity.
To address specificity and sensitivity three optimal tumor associated antigens specific toward pancreatic adenocarcinoma were chosen: CEA, Ca 19-9, and CAM 17-1 (Diamandis, 2002). With a reduced fluid volume, sensitivity and specificity are important as only a few molecules are tested with this system leaving little room for error. The accuracy of a LOC device diagnosis can only be assured if multiple antigens are tested for in the microfluidic assay to decrease probability of a false match. After filtration and concentration of the analyte, the probe antigen adsorb through the nanochannel walls binding to its Bovine Serum Albumin (BSA) functionalized surface (Pereira et al, 2011). Using a syringe, phosphate buffered saline (PBS) was flowed through the input of the system to wash off any weakly bound antigens on the channel walls. The immunoassay is then performed as the immobilized antigens come into contact with their primary antibodies. CEA was allowed exposure to anti-CEA mouse monoclonal antibody. While CEA was the sole method of serological detection for pancreatic cancer for over a decade, it is not particularly sensitive. As such Ca 19-9 is used to increase the sensitivity and specificity of the immunoassay. Alone, this antigen-antibody reaction results in a sensitivity of up to 93% and a specificity of up to 98% excepting Lewis-negative individuals. To account for Lewis-negative phenotypes and to add further specificity mucin 17 is tested against monoclonal antimucin antibody CAM 17-1 (Diamandis, 2002).
Secondary antibody complexes were formed with Invitrogen goat polyclonal anti IGG conjugates tagged with three quantum dots of differing wavelengths for each primary antibody-antigen complex. Upon the bound CEA -anti CEA mouse monoclonal antigen-antibody complex the injected goat anti mouse IgG secondary antigen will attach itself allowing the QD to emit light at 625nm (Invitrogen; Carlsbad, California). This emission will be detected by the photodiode (Pereira et al, 2011). Following upon above procedure, at the binding of rabbit mononoclonal Ca 19-9 to 1116 NS 19-9 (IgGl) (19-9-Ab) goat anti-rabbit IGg will allow the 705nm QD to emit (Invitrogen; Carlsbad, California). Finally, the binding of rat anti mucin CAM 17-1 to polyclonal goat anti-rat allows the QD to emit at 565nm (Invitrogen; Carlsbad, California). The emission of all three wavelengths indicates a highly sensitive and specific positive test result.
Mechanics and Functional Analysis:
Once the LOC and photodiode have been manufactured, diagnostic immunoassay testing can begin. The sample to be analyzed is first injected into the fluid input of the chip. Once injected, the sample is filtered by the TEMs, thereby removing any large proteins and other biologics larger than 15nm. The TEMs filter also serves to encourage even fluid flow. (Ferain et al, 1997) The filtered sample is then left to partially absorb into the PDMS nanochannels for approximately 10 minutes. Next, the chip is flushed with a washing solution of bovine serum albumin (BSA) dissolved in phosphate buffered saline (PBS) for approximately 3 minutes. This washing procedure serves two functions: 1) to remove any analytes weakly bound to the nanochannel surface and 2) to deposit a layer of BSA onto the nanochannel surface anywhere where there has been no analyte absorption. (Pereira et al, 2011) The BSA will serve as a surface blocking agent, which is critical to the prevention of a false positive as it prevents the primary and secondary antibodies to the analyte being tested for from being absorbed into the PDMS nanochannels. After the chip has been flushed, the chip is now ready to receive an injection of primary antibodies specific to the antigens being tested for. The antibodies are dissolved in PBS. The chip is left to sit for another 10 minutes to allow for the binding of the primary antibodies to the analytes. (Pereira et al, 2011) Once the primary antibodies have had sufficient time to bind their antigens, the system is again flushed with PBS. The system is now ready to receive an injection of secondary antibodies (also dissolved in PBS) that recognize the primary antibodies already bound to the analytes being tested for, assuming that the analytes being tested for are indeed present. These secondary antibodies serve as an immunoflourescent agent are what allow for the detection of the analytes, as each type of secondary antibody is bound to a specific quantum dot (Qdot). Different Qdots are excitable at different wavelengths of light, thereby making it possible to differentiate between the secondary antibodies present during analysis based upon the light emission observed for each excitation wavelength exposed to the chip. (Giessen and Lippitz, 2010) The chip is again left to sit for 10 minutes to allow for the binding of the secondary antibodies to the primary antibodies. Once the secondary antibodies have had sufficient time to bind their primary antibody counterparts, the chip is once again flushed with PBS so as to remove any free secondary antibodies. This is an extremely important step due to the fact that if the chip were not flushed after the introduction of the secondary antibody-Qdot complexes, any unbound secondary antibodies would remain present in the nanochannels and could potentially produce a false positive during analysis. Once this final PBS flush has taken place, the chip is ready for analysis. (Pereira et al, 2011) Important to note is that due to large fluid flow impedance at the nanoscale, flow of all fluids after injection into the chip is assisted via electrostatic exclusion resulting from an unbalanced cationic flux. This flux is generated through an electric field, which is applied at the interface between the TEMs and the nanochannel via thin film gold electrodes. (Kovarik et al, 2009).
Analysis of the prepared chip is relatively simple. An optical filter is utilized to block light at any of the excitation wavelengths from reaching the chip, except for that light purposefully applied to the chip. This allows for the more precise and accurate detection of each Qdot type present. Once the filter is in place, a fluorescence excitation laser beam that has been calibrated to emit light at the specific excitation frequency of one of the Qdots used in the secondary antibody-Qdot complexes is directed at the chip. (Pereira et al, 2011) This procedure is then repeated for each Qdot excitation frequency. If any Qdots that are excited by the frequency of light being exposed to the chip are present, they will fluoresce at a very specific wavelength and can readily be detected by the incorporated photodiode. (Giessen and Lippitz, 2010) Emission light intensity provides information about Qdot concentration in the chip, which in turn provides information on the concentration of analyte being tested for. This information can be used to determine not only the results of the test, but also potentially the stage of the condition being tested for, as later stage cases should have higher concentrations of analyte present in blood samples.
Using this methodology, we propose the creation of a LOC for the early detection of pancreatic cancer. To do so, first a number of antigens indicative of pancreatic cancer must be identified. We will be looking for the pancreatic cancer tumor associated antigens CEA, Ca 19-9, and CAM 17-1. (Hoff, Evans, and Hruban, 2005; Diamandis et al, 2002) Three antigens are being tested for as with each additional antigen tested for, the specificity and sensitivity of the test is increased. Test antigens identified, monoclonal antibodies recognizing these antigens must either be identified or created. These monoclonal antibodies will serve at the primary antibodies discussed above. Secondary antibodies that can recognize the primary antibodies with sufficient specificity must then either be identified or created as well. Luckily, monoclonal antibodies to these antigens are already known and therefore do not have to be synthesized. For our primary antibodies we will use anti-CEA mouse monoclonal antibody for CEA detection, 1116-NS-19-9 (IgG1) (19-9 Ab) rabbit monoclonal antibody for Ca 19-9 detection, and rat anti-mucin antibody CAM 17-1 for CAM 17-1 detection. The secondary antibodies recognizing our primary antibodies that we will use are goat polyclonal anti-mouse IgG for CEA primary antibody detection, goat polyclonal anti-rabbit IgG for Ca 19-9 primary antibody detection, and goat polyclonal anti-rat for CAM 17-1 primary antibody detection. (Hoff, Evans, and Hruban, 2005; Diamandis et al, 2002) Each type of secondary antibody will have its own unique Qdot that fluoresces at its own unique frequency: a 625nm wavelength emission Qdot for the CEA secondary antibody, a 705nm wavelength emission Qdot for the Ca 19-9 secondary antibody, and a 565nm wavelength emission Qdot for the CAM 17-1 secondary antibody are proposed. (Invitrogen, Carlsbad, CA) The emission of all three wavelengths indicates a highly sensitive and specific positive test result.
Methodology for Self Assembly and Nanofluidics:
As technology advances from micro to nanoscale the need for structural organization is crucial for system function. Further, due to the small scale order of these systems as well as the in vivo applications human or even computer guidance is not feasible. As a result, self-assembling systems are essential when using LOC and other nanoscale procedures. Self-assembly (SA) is the process in which no external direction is administered and pre-existing disordered parts assemble by means of local interaction to form a structure or pattern (Whitestone 2001,2002; Philip 1996). There are two types of SA, either static or dynamic. Static assembly occurs when the system approaches or sustains equilibrium and does not lose energy. Contrary to static, dynamic assembly is more of a self-organizing system and requires the dissipation of energy (Whitestone 2001).
The SA involved in LOC is slightly more complex because it is the intermolecular assembly of molecules with the final goal of coding components to form a desired shape and exhibit specific properties for the nanosystem. Many systems are deceiving and appear to undergo SA due to the increase in internal system organization without external guidance. However, SA consists of several distinct concepts: individual components, interactions, and directive(Whitestone 2001; Philip 1996). Individual components must span a range of nanoscale building blocks (NBBs) and cannot simply be atoms or only molecules. The system depends on a variety of functioning meso and nanoscopic structures to allow for different chemical properties, operation, and shape. The second concept, interactions, heavily relies on the weak interactions as oppose to covalent or ionic bonds. These weak bonds consisting of Van der Waals, pi-pi, hydrogen, and capillary bonds play a key role in the existence of interim attraction and inclusive repulsive forces that effect molecular organization in membrane systems. Lastly, the total assembled system must have higher order then each of its individual components. This proves opposite when compared to most chemical reactions but the disordered state must proceed towards order for a fully functional and useful LOC (Whitestone 2001,2002; Philip 1996).
Although SA is vital in LOC it is a fundamental principle and further examination of the molecular processes that lead to SA is necessary in understanding the system. The question is what is the main function of the LOC system? The system depends on flow rate (Q), flow time (t), concentration (C) and volume (V) resulting in two equations, Q = V/t and C = n/V. These factors then determine velocity (vo), desired height or diameter (h, d), and regime time (diffusion tD and convection tC) (Gleeson 2004; Lee 2008; Pereira 2011). Our design depends on antibody absorption so the design would be based on optimum criteria to promote antibody binding. In total, each of these factors must be chosen to optimize antibody binding and will influence the Peclet number which defines the system. Peclet number (Pe) is the ratio of the distinctive time scales for advection and diffusion, Pe = h vo /D where D is the mass diffusion coefficient. It can be further explained as a ratio of the rate of transport of a substance in a particular direction by the flow to the rate of a gradient driven diffusion, or relates tD and tC due to vo (Gleeson 2004; Lee 2008; Pereira 2011; Weddemann 2009)
When Pe is large ('>10) axial velocity interacts with the cross sectional transverse diffusion resulting in a convection dominated regime (Adrover 2009; Pereira 2011). This will result in molecular flow through the length of the channel and little time for diffusion across the wall. Since we want to favor absorption of antibodies on the wall and diffusion across the channel a small Pe is required. To further influence antibody absorption and antibody-antigen reactions in the system tD and tC should be on the same order with a slightly higher tD to favor diffusion (Adrover 2009; Lee 2008; Pereira 2011). In previous literature various numbers were used for Q, C, V, h, tD, tC and vo. Without testing each of these values the most effiecient value will remain unknown. However, using previous experiments and studying results an estimate for each variable can be assigned with confidence. Our design will use a Q = 6.8 nL/min, a conventional antibody diffusion coefficient of 4E-11 m2/s, C varying between 10 ' 1000 mg/L, tD ~ 1 and tC ~ .8 (Lee 2008; Pereira 2011). The exact dimensions will be determined with further research but generally a LOC's length is around 100'm-300 'm, a height of 20 - 70'm, with subsequent flow velocity of 100'm/s to as high as 5cm/s (Weddemann 2009, Pereira 2011). Since a smaller Pe value of 5 ' 7.5 is desired the h and vo can be determined by setting one and solving for the other. A height of 20 'm yields desirable results which would produce a flow velocity of 13'm/s.