Keywords

Introduction

Novel nanobiosensors have the potential to vastly improve current standards and techniques for the diagnosis and treatment of cancer. Many nanobiosensor systems have proven remarkably successful in research models of various cancers and the future promises further translation of these nanomaterials and techniques into clinical practice. A number of advantages compared to traditional biosensor systems make nanobiosensors attractive modalities for development. Detection sensitivity can be dramatically increased due to the nano-sized components utilized in the construction of nanobiosensors. It is feasible to engineer smaller sensors that can be used to decrease lab space requirements or possibly create implantable monitors. Enlargement of the operative range is possible through extension of upper and lower limits of detection for biomarkers. Finally, fabrication costs of these nanosensors can be decreased using advanced technology and this can in turn decrease clinical healthcare testing expenditures for the detection and diagnosis of cancer.

A major reason why cancer continues to cause mortality among patients is that it is not detected early enough and current treatments for advanced stages are not as effective as treatment protocols for cancer that is caught early [1]. According to the American Cancer Society, cancer is the second leading killer across all age groups, behind cardiovascular disease, and in 2012 it is expected that 577,190 Americans will die from cancer [2]. It is promising to note that recent progress has been made in survival from cancer as evidenced from an increase in survival rate. The most current available data documenting survival from cancer during the time period from 2001 to 2007 is 67 %, which is significantly higher than the 49 % from the period of 1975–1977 [2]. The trend of increasing survival rates from all cancers has been attributed to technology that has improved treatment options and diagnostics [3]. Although the general trend is an increased cancer survival rate, there are still many types of cancer that remain quite deadly, particularly when diagnosed in a later state (pancreatic, liver, lung, etc.). Thus, one of the main issues that has limited adequate treatment of cancer is not detecting it early enough and as the cancer progresses it becomes increasingly difficult to treat [4]. Further, approximately 60 % of cancers are diagnosed after the patient’s initial tumor has metastasized [3]. Developing and implementing new nanobiosensors for use in the clinical setting has the potential to vastly improve early noninvasive diagnostics, enhance imaging, and advance monitoring of treatment progress.

In order to accurately detect evidence of cancer in a biological sample, normal specimens must be differentiated from that which is cancerous. To accomplish this, various biomarkers are employed. A biomarker is a molecule that can be found in tissue specimens, blood, exhaled breath, or other fluids and can indicate a disease or health status [3]. More specifically, biomarkers can be alterations of cellular DNA, RNA, proteins, or metabolites that helps distinguish between health and disease as well as allow us to monitor treatment progress [5]. Knowledge of cancer biomarkers continues to become more sophisticated for early cancer detection and determination of prognosis. A discussion of important biomarkers currently being integrated into nanobiosensor technologies will be reviewed in later sections.

The field of biosensors has been around for nearly four decades [6] and recently much academic research has been invested in experimenting and developing new methods using nanotechnology that promise to improve current standards of disease detection. The field of nanobiosensors especially in relation to cancer detection is still only in its infancy but new technological advancements and further development in understanding the complexities associated with cancer behavior should help to improve treatment outcomes by driving down many of the morbidities and mortality rates associated with cancer.

Nanobiosensors work at the level of the nanoscale broadly defined as between 1 and 500 nm or roughly 1 billionth of a meter. Development of nanobiosensors has been most successfully achieved through multidisciplinary collaborations encompassing the fields of medicine, biology, chemistry, physics, engineering, and technology. The unique contribution that nanotechnology has made are possible due to increased knowledge in the molecular biology field and the ability to exploit special properties of materials at the nanoscale through engineering and material science [7]. There are two main reasons materials have unique properties at these small sizes: (1) there is a large surface area to volume ratio which means that many of the atoms that comprise the material are a close distance from the surface of the material; (2) quantum forces are exhibited due to the fact that the material size is close to wavelengths which can excite the nanomaterial components [7].

This chapter takes the approach of first describing the characteristics known about a select number of modalities and components that comprise nanobiosensors. Particular emphasis is placed on nanoparticles, nanowires, carbon nanotubes, and cantilevers (Fig. 1). Then examples of the above-mentioned nanobiosensors are illustrated from the recent literature in the areas of diagnosis, detection and imaging of prostate, breast, pancreatic, lung, and brain cancers.

Selected Modalities

Nanoparticle s: In many nanobiosensor systems, nanoparticles often play a crucial component for detecting different cancers. These particles can be made of a variety of materials and the unique properties that each possess have been engineered and exploited to achieve enhanced biomarker detection. In general, nanoparticles function in various ways to provide clinical utility whether it be through diagnostic applications such as imaging or biomarker detection, therapy application, or a combination of diagnostic and therapeutic also referred to as theranostics [8]. The first generation of nanoparticles in oncology research were simple in design and functioned as either a therapeutic cargo delivery method by allowing improved delivery of drugs to cancer sites or as an imaging agent for tumors. As research progressed in the nanotechnology field, so did the sophistication of nanoparticles. The most recent nanoparticle platforms incorporate diagnostic and imaging functions simultaneously with surface functionalized nanoparticles for targeted delivery and increased uptake at the desired area. The following sections highlight some important general categories of nanoparticles used in cancer diagnostics as well as discussions of properties that make them advantageous constituents of many nanobiosensor systems. As you will see, many of the properties of one type of nanoparticle can be applied to others.

Quantum dots: Quantum dots (QDs) are one category of nanoparticles that have been used for both in vitro and in vivo cancer detection. These are nanocrystals that are made up of semiconductor particles, consisting of an inorganic element in its core with a surrounding metal shell [9]. They generally measure less than 10 nm in diameter [10]. The benefits of using QDs in research, diagnosis, and treatment of cancer are due to some of their unique properties. The first of these properties is that their size and composition can be adjusted to give the QDs a unique fluorescence emission that can vary from 400 to 2000 nm [9, 11]. Varying wavelengths allow for tuning QDs to any color, which enables recognition and tracking of differently labeled biomarkers using only a single light source [12]. Another characteristic that makes QDs useful is their resistance to photobleaching and thus they can be used for an extended period of time [9]. One problem that is traditionally seen with imaging normal healthy tissue is that it often exhibits autofluorescence and this interferes with the signal from cancerous tissue. QDs have been engineered to have fluorescence properties in the near-infrared spectra and thus can eliminate much of this interference [9, 13]. A potential problem involved in using QDs in vivo is whether injection poses a toxic risk or not. Modifications have been made to decrease potential toxicity, however, more research needs to be carried out to determine appropriate clinical adaptability [14].

Paramagnetic nanoparticles: Contrast agents are an important consideration for detection of tumors in any region of the body. Superparamagnetic iron oxide nanoparticles (SPIONs) have been used as contrast agents in imaging cancer tumors with both magnetic resonance imaging (MRI) and computed tomography (CT) and are the most established nanomaterial in clinical practices [15]. These agents can vary in size from 50 to 100 nm for some particles to an even smaller size range of 5 to 10 nm for other particles [16]. Generally speaking, particles with smaller size tend to be taken up by a wider range of tissues including lymph nodes and bone marrow. SPIONs can also be functionalized with antibodies to various tumor markers to improve uptake and imaging procedures [17]. It has been shown through certain functionalized surface modifications, these nanoparticles can differentiate between non-apoptotic and apoptotic tumor cells. This knowledge could help guide clinicians in determining the most prudent chemotherapeutic regimen for a particular patient through easily and directly monitoring response to treatments [18]. Magnets located external to the body surface can be used to exploit intrinsic magnetic properties of these nanoparticles by directing them to a tumor site. This targeting mechanism can be coupled with cytotoxic drugs encapsulated within the nanoparticle to greatly increase the concentration of drugs at tumor sites. The goal of this therapy is to decrease the total amount of drug being administered which in turn minimizes many potential toxic side effects patients must tolerate while being able to monitor this process through MRI [19].

Nanoshell s: Nanoshells comprise another category of nanoscale particles that combines a dielectric core with a surrounding metal shell often composed of gold. These particles are particularly useful due to their ability to be tuned to different optical resonances by adjusting the size of the core and shell similar to the QDs mentioned earlier [20]. Nanoshells extend their effectiveness by incorporating the ability to be used as an imaging component in addition to having the ability to load drugs into the core [21]. Antibodies have also been conjugated to nanoshells allowing for targeted delivery of its cargo to specific tumor types [22]. One common means of increasing circulation half-life of nanoshells and other nanoparticles is coating the outer surface of the nanoshell with poly ethylene glycol (PEG) otherwise known as PEGylation. In addition to increasing in vivo circulation, PEGylation has also been shown to decrease toxicity of gold nanoshells, decrease aggregation, improve avoidance of macrophages, and the reticuloendothelial system [23]. Another interesting application of nanoshells that has been reported is the ability to generate heat after exposure to near-infrared light [24]. Heat generated at the tumor site where nanoshell have aggregated can cause a photothermal ablation effect to kill cancer cells [25]. In this way nanoshells can have a diagnostic imaging function combined with cytotoxic consequences.

Gold nanoparticle s: Another class of particles which have shown promise in diagnostics is nanoparticles made from colloidal gold. Gold nanoparticles mostly exist as gold nanospheres, which exhibit an intense ruby color in aqueous solutions [26]. The fascinating optical properties of gold nanoparticles arise from localized surface plasmon resonance (LSPR), in which valence electrons in gold nanoparticles oscillate coherently with incident light at specific frequency [27]. Part of the energy absorbed by gold nanoparticles is emitted in the form of scattered light, which forms the basis of gold nanoparticle-based optical imaging [28]. The rest of the energy decays in a nonradiative form which is converted into heat, which can be used in killing cancerous cells and hence play a role as photothermal therapy. Gold nanoparticles may find use in multiple diagnostic potential areas such as labeling precancerous cervical biopsies [29] as well as other prospective uses in cancer treatment [30].

Liposome s: Liposomes are nanoparticles composed of a lipid bilayer which can be used in transporting imaging agents or drugs to the site of a tumor. Along with other types of nanoparticles, liposomes use the property of enhanced permeability and retention (EPR) to enter tumor vasculature and remain there. The phenomenon of EPR stems from a structural understanding of vasculature associated with solid tumors. Nanoparticles have increased ability to extravasate through less tightly formed endothelial junctions. Once in the tumor microenvironment, increased retention is due to inadequate lymphatic drainage [31]. In order to utilize the EPR effect, liposomes are usually engineered with a size of less then 200 nm to improve accumulation in the tumor [32]. Liposomes can also be labeled with a targeting component in order to increase cellular uptake at target tissue.

Nanowire s: Nanowires also make up an interesting category of biosensors. A nanowire can be defined as a material consisting of millimeters in length but achieves a diameter that is measured in the nanometer range. Nanowires composed of silicon have been best characterized [33] due to advancement of manufacturing techniques at the large scale and represent an example that may prove useful as point of care diagnostic devices [34]. Their ability to detect biomarkers in a sample, such as a drop of blood, rely on the nanowire being a field effect transistor (FET) device. The basic concept behind biosensing in this application is when a biomolecule of interest comes into contact with the nanowire, a measurable change in the electric field of the nanowire occurs due to increased resistance. Further, direct detection utilizing nanowires has been show to be extremely sensitive for a number of different substances including: metal ions, nucleic acids, proteins, protein-DNA interactions, small molecule–protein interactions, cells, and viruses by surface functionalization techniques [34]. Specific applications will be mentioned in the following sections regarding how nanowires can be used to detect cancer biomarkers.

Carbon nanotube s: There has been much interest in the area of carbon nanotubes (CNTs) since their discovery over 20 years ago by Japanese physicist Iijima [35]. Carbon nanotubes are structures composed of either single-walled or double-walled carbon molecules self-arranged in hexagon pattern with diameters ranging from 0.3 to 100 nm [36]. There are several characteristics that make CNTs attractive for use in nanobiosensors. High heat resistance and conductivity allow CNTs to remain operable even while exposed to high temperature or electrical currents. CNTs also exhibit a high degree of tensile strength that permits them to resist permanent deformation after exertion from physical forces. Often CNTs are placed into two general categories based upon how they sense biomarkers, they can generate an electrochemical signal based on oxidation–reduction reactions that occur or as FET-based detection which derives its signal from charges generated on the carbon nanotube surface [37] similar to nanowires described above. Nanotubes of both single and multiple-walled types have been used in an application known as nanotube forests. This technique requires nanotubes to be lined up next to each other in a parallel fashion and then antibodies for specific biomarkers are attached to the surface. After the binding interaction of antigen and antibody, an additional binding event can occur between an antibody coupled to a signaling agent which binds the other side of the antigen and essentially makes a sandwich. Measurement of the amount of antibody binding events allows for quantification of cancer biomarkers. The other common method of detection incorporating carbon nanotubes is the previously mentioned FET method, in which a nanotube is connected to an electrode at either end using photolithography and functionalized to be able to bind biomarkers. Once these biomarkers are bound, a decrease in conductive properties of the nanotube is detected that is proportional to the amount of binding that has occurred.

Nanocantilever s: The last general category of nanobiosensors that will be discussed in this paper is nanocantilever devices. Cantilevers are able to conduct biosensing through the principle that these tiny probes naturally vibrate at a certain frequency dictated by mechanical and mass properties. When a biological molecule binds to this nanoscale probe it will alter baseline probe frequency, this change is typically measured by a difference in the characteristics of the light deflection pattern of the probe or through electrical means [38]. Different nanocantilevers systems can detect different phenomenon regarding behavior of the probe. Two common types of probes exist: static, which when bound by a biological molecule will bend to one side or another, and resonant probes, which when bound by a biological molecule change their resonance frequency. Interestingly, it has been observed that cantilevers with diameter in the micrometer or thicker range tend to decrease resonant frequency when attaching mass while nanosize cantilevers tend to increase frequency when mass such as antibodies are attached. This is explained by effecting a property called the net stiffness constant of the nanocatilever as mass is added [38]. Nanocantilevers have been described as a simple replacement to PCR reactions and detection methods because they do not require costs associated with sample preparation such as time and expensive materials. Nanocantilevers could also be used to monitor various cancer biomarkers and expand the usefulness of microarray technologies [39].

Applications in Various Cancers

Prostate Cancer

The PSA screening test is the most widely used screening tool for prostate cancer and the long controversy surrounding it illustrates the need for more sensitive and specific tests that can better determine a patient’s course of treatment [40]. Prostate-specific antigen (PSA) is a glycoprotein secreted by epithelial cells of the prostate gland that was first discovered and utilized for forensic purposes [41]. In 1994, the Food and Drug Administration approved the PSA test for prostate cancer screening in men over 50 along with digital rectal examination and this was the standard screening protocol for years [42]. Most recently, in May 2012, the U.S. Preventative Services Task Force (USPSTF) recommended against the use of the PSA screening test [43]. The USPSTF’s report highlights the need for a better diagnostic test that can distinguish between aggressive cancer and types of cancer that are nonprogressive or progressing so slowly that it won’t affect the patient’s life [43]. Current work in nanobiosensors and tumor marker discovery may help to create and validate such a test that can spare patients unnecessary procedures and biopsies if they are unlikely to benefit.

Nanoparticle s: Although the PSA test has been scrutinized for its clinical utility as a general screening test, it has been accepted as an important tool in monitoring the status of cancer recurrence after radical prostatectomy as well as determining response to treatments. One emerging nanotechnology assay technique that may prove useful clinically for PSA testing is bio-barcodes [44]. These bio-barcodes have been shown to be hundreds of times more sensitive than conventional assays used commercially [45]. The concept behind bio-barcodes is that attached to gold nanoparticles are antibodies to PSA and hundreds of strands of DNA barcodes. Magnetic bead particles also have PSA antibodies attached. When PSA antibodies of the magnetic beads and gold nanoparticles encounter the PSA glycoprotein they form a complex by sandwiching the PSA molecule between them. A magnetic field is then used to separate the complex from the sample. Subsequently, the amount of DNA barcode that has been separated can be quantified to determine the amount of PSA in the sample [45, 46].

Detection of cancer cells in blood can also serve as an indicator of a patient’s cancer status. Prostate cancer cells can be tagged with gold nanoparticles so that the cells can be detected in blood through a photoacoustic flowmeter. Through this method cancer cells can be detected, making it have great potential as a serum detection method [47]. Another important detection modality that utilizes gold nanoparticles is to detect whether prostate cancer cells have migrated to lymph nodes. In order to determine if migration has occurred, highly lymphotropic superparamagnetic nanoparticles have been shown as effective clinical imaging agents to detect small metastasis in lymph nodes through MRI [48].

Nanoparticles that fall into the category of theranostic nanoparticles may play a future role in prostate cancer treatment. These multifunctional nanoparticles allows for simultaneous administration of therapeutic and diagnostic agents and can even be taken a step further by having a targeting agent attached to the outer portion. This addition helps to improve nanoparticle cargo uptake at target cancer cells [49]. These nanobiosensors hold promise for improved detection and survival for patients with prostate cancer, whether it be on initial diagnosis, monitoring, or planning the most effective treatments.

Nanowire s: Another category of nanobiosensors that may prove beneficial for prostate cancer patients is nanowires. Silicon nanowires have been reported to be able to detect PSA at the level of 1 fg/ml of PSA through optimization of dimensions of the nanowire as well as amount of doping concentrations for antibody functionalization during nanosensor construction [50]. Further, multiplexed tumor antigen recognition is possible utilizing silicon nanowires. Zheng et al. were able to construct arrays composed of nanowires functionalized with antibodies against PSA, PSA-alpha1-antichymotrypsin, carcinoembryonic antigen, and mucin-1. They were able to reliably detect these biomarkers at femtomolar concentrations in undiluted serum samples [51]. Another system reported by Stern et al. uses a two-step approach that incorporates microfluidic purification chips which capture multiple biomarkers from whole blood, concentrates the analyte with the biomarkers of interest, and releases the biomarkers for quantitative detection with silicon nanoribbon detectors. This technique reduces the minimum required sensitivity of the system [52].

Carbon Nanotubes: Carbon nanotubes forests have been shown to present advantages for cancer antigen detection. One group reported up to 15-fold increased detection sensitivity of cancer antigens based on their ability to densely pack and immobilize more surface antibodies compared with sensors utilizing a flat surface [53]. The proteins: PSA, prostate-specific membrane antigen, platelet factor-4, and interleukin-6, all known to be increased in prostate cancer patient serum, were measured simultaneously through an array based on carbon nanotube forests [54]. Examples of carbon nanotube forests are increasing in the literature and represent important assays that can detect multiple antigens during one measurement with important implications for better identification and improved risk stratification for cancer patients.

Nanocantilvers: PSA antigen antibody binding has also been detected using piezoeclectric nanomechanical cantilevers. Analysis of one type of cantilever used in PSA detection showed resonance frequency change as a result of surface stress placed on the nanocantiliver rather than from increase in protein mass associated with binding events [55]. Some reported benefits of using nanocantilever systems in cancer detection are there are no requirements for fluorescent or radioactive labeling, detection can take place in liquid samples, and this technology can easily be translated to lab on a chip techniques providing point of care diagnostics [56].

Breast Cancer

Although there have been many advances in the detection and treatment of breast cancer, it still remains the most prevalent cancer in women with about 230,480 women being diagnosed with breast cancer in 2011, and almost 40,000 deaths [57]. Current strategies for treating and diagnosing women with breast cancer often carry noteworthy side effects, require invasive procedures and leave women permanently scarred [58]. The application of nanobiosensors has been applied to multiple facets of breast cancer diagnosis. The areas that will be discussed in the following paragraphs are early detection methods, improved prognosis estimation, advances in sentinel lymph node biopsies, screening, and monitoring treatment responses in breast cancer.

One biomarker that has provided both diagnostic and therapeutic usefulness in breast cancer is HER2, which is a plasma membrane-bound receptor tyrosine kinase encoded by the ERBB2 gene [59]. Amplification of this gene occurs in about 30 % of breast cancer patients and when detected indicates a worse prognosis [17].

Nanoparticles: There have been a numerous examples of nanoparticle systems utilizing HER2 introduced for detection of breast cancer. Wu et al. showed that attachment of IgG and streptavidin to QDs can be used to fluorescently label HER2 positive cells on both histologically fixed cells as well as live cells. The benefit of using QDs over traditional staining procedures is that with QDs the authors were able to show a stronger targeted fluorescent signal that was more stable than traditional techniques. In addition, this form of labeling can be used for multiple cellular targets and simultaneously excite various colored QDs with only one light source [60]. Similarly, a study done by Chen et al. showed that their QDs-based probe was more accurate and sensitive than immunohistochemical techniques in detecting HER2 in clinical breast cancer samples [61]. Other fluorescently labeled silica nanoparticles have also shown a high degree of sensitivity to breast cancer cells when conjugating anti-HER2/neu to the nanoparticle [62].

Multiple examples in the literature demonstrate the use of superparamagnetic nanoparticles conjugated to various targeting moieties to serve as targeted contrast agents in MR imaging. The HER2/neu receptor has been used as a target for streptavidin conjugated superparamagnetic nanoparticles made from iron oxide. The nanoparticles in this instance were imaged using MR molecular imaging and proved to have proportional contrast with the amount of HER/neu expression on the surface of the cells that were being studied [17]. LHRH conjugated to SPIONs have shown utility through a mouse model for in vivo detection of lymph node metastasis along with MRI [63]. In addition, SPIONs have been functionalized with a recombinant peptide that targets urokinase-type plasminogen activator (uPA) receptor known to be overexpressed in breast cancer tissues [64].

Herceptin, the antibody that targets HER2, has been attached to gold nanoparticles so that the nanoparticles could be used as a targeted contrast agent while using the imaging method of optoacoustic tomography. The authors of this study were able to show that using this method in a gelatin breast model, it was possible to sensitively detect small tumors that were implanted up to 6 cm deep [65]. Herceptin conjugated to iron oxide nanoparticles was also tested using ultrasound as the imaging modality and was found to be able to elicit a significant ultrasound signal and thus has the potential to be used in the future as an inexpensive detection method for breast cancer [66].

Another strategy incorporates the use of ultrasound for both diagnostic purposes and therapeutic delivery of drugs. Two different nanoparticles were used in this study: polymeric micelles loaded with doxorubicin and echogenic nanobubbles loaded with doxorubicin in the walls of the nanobubble. The polymeric micelles use the well-known EPR effect that is seen in all tumor vasculature for delivery to the tumor site. The nanobubble also utilize the EPR and once at the tumor site the nanobubbles coalesce into a microbubble. This allows for increased drug delivery from the nanobubble, increased drug delivery from the corresponding micelle and also a high quality contrast agent for ultrasound imaging. In addition, the ultrasound can be adjusted to more precisely control where the drug in the nanobubbles is released [67].

Gold nanoparticles have been functionalized with a variety of surface targeting molecules such as transferrin. The transferrin receptor is important for iron uptake in normal cells and cancers. Tumor cells are in a stage of rapid proliferation so they up regulate transferrin receptors to maintain growth [68]. It has been shown that transferrin-mediated cellular uptake of gold nanoparticles is more pronounced by a factor of 6 in breast cancer compared to non-cancerous cells [69].

Another class of nanoparticles that uses HER2 as a target are nanoshells. Nanoshells are particles that are made of a dielectric core and covered with a gold shell. These particles can be adjusted similar to QD to have different optical imaging properties and absorb near-infrared (NIR) light [70]. Nanoshells have added benefit because they have also been shown to have dual therapeutic and diagnostic imaging properties. They are able to reflect light and thus be used in imaging as well as absorb light allowing the nanoshells that have been taken up in the cells to increase in thermal activity thereby killing breast cancer cells [22].

In addition to the potential use of nanoparticles for detection of primary breast cancers that have remained localized, nanoparticles have also shown promise in assisting surgeons in identifying breast cancer that has metastasized to the lymph nodes. One challenge that surgeons face when performing a masectomy after detection of breast cancer is determining the extent, if any, the primary cancer has spread to lymph nodes [71]. Sentinel lymph node mapping has greatly improved surgical treatment of women with breast cancer due to less nodes having to be removed for examination by the surgical pathologist which in turn results in a more concentrated histological examination and decreased morbidity [72]. The current clinical standard generally involves the surgeon taking one of the two approaches, either injecting vital blue dyes or using radioactive technetium-99 m sulfur colloid [73]. Both ways present drawbacks due to radioactive exposure to patients or limited visibility using dyes. QDs and ICG-human serum albumin nanoparticles have been proposed as mitigating the above problems and providing improved intraoperative imaging of sentinel lymph nodes using fluorescence imaging systems that utilize NIR wavelengths and improved contrast compared to current approaches [74, 75]. A study by Ballou et al. has shown that PEG coated QDs could be used as a method for identifying sentinel lymph nodes in mice [76]. Again, in other studies, single molecule targeting is used and sentinel lymph nodes are detected using QDs [77, 78].

Sentinel lymph node mapping can also be done using gold nanotubes. The benefits of one reported method is that it uses photoacoustic imaging so it is noninvasive and can be used to sensitively detect sentinel lymph nodes in tissues that are up to 33 cm deep [79].

Another interesting area that is currently being researched is in vivo characterization of tumor subtypes so that specific treatment protocols can be rapidly implemented and carried out. Again, QDs have been used to study and differentiate cancer subcategories by using different markers in a single tissue specimen [80]. In addition, dendrimer nanoparticles have been created which can be dually utilized for imaging using MRI or NIR fluorescent modalities in the single probe. This has been shown effective in mouse sentinel lymph node mapping as well [81, 82].

One common way of obtaining a biopsy of a breast lump is through fine needle aspiration (FNA). Issues can arise using FNA as a diagnostic test for cancer due to the high amount of false negatives and false positives originating at least partially from the limited biopsy sample of this method. Nanobiosensor techniques are ideal for the specific difficulties of analyzing the FNA sample. Lee et al. devised a method that can label breast cancer cells with magnetic nanoparticles that have been obtained through a fine needle aspiration. They then used a diagnostic magnetic resonance probe to detect characteristics often seen in cancer cells as compared to normal non-cancerous cells. This method illustrates the ability to maintain a sensitive test while only requiring a small amount of tissue from the fine needle aspirate [83].

Nanowires: One of the capabilities of nanowires is to detect protein-DNA interactions. An illustration of the importance of using this interaction for breast cancer detection can be seen with an example from the estrogen receptor (ER). The ER plays an important role in many normal physiological processes but an abnormal ERalpha exists in over half of breast cancers where it is a ligand-dependent transcription factor and plays a role in cancer initiation and progression [84]. A sensitive assay for detection of ERalpha interaction with double-stranded (ds)DNA has been developed. In this assay a silicon nanowire was surface functionalized with dsDNA of wild-type estrogen receptor elements. When nuclear extracts of breast cancer cells expressing the ERalpha receptor were evaluated using the nanowire apparatus there was notable detection due to abnormal receptor binding to dsDNA functionalized nanowire and this was distinguishable from normal ER binding. The conductance change reported for this nanowire was able to detect a biomarker protein concentration as low as 10 fM [85].

Carbon nanotubes: The use of carbon nanotubes has been demonstrated in a number of different purposes for detection of breast cancer. Using carbon nanotubes functionalized with IGF1R and Her2 antibodies (known to be upregulated in some breast cancer cells) one group created a device able to detect single circulating cancer cells from human breast cancer cell lines MCF7 and BT474 in two microliter drops of blood [86]. A group used paclitaxel (PTX) as a therapeutic agent and coupled it via a cleavable ester bond to the PEGylated nanotube surface. This construct was tested in a murine 4T1 breast cancer model and the results depicted a 10-fold increase in tumor accumulation than PTX alone followed by improved tumor suppression compared with clinically used Taxol [87]. They also reported the coupling of other agents like Pt(IV) prodrug PEGylated carbon nanotubes to improve the pharmacokinetics and therapeutic effects [88].

Nanocantilevers: Detection of BRCA1 mutations can be an important tool in determining risk for development of breast cancer, ovarian cancer, and prostate cancer. A cantilever assay has been developed that can detect a single nucleotide polymorphism (SNP) for the BRCA1 gene. The benefits of this assay is that it is highly specific and it only takes about 30 min to complete the process of DNA immobilization of the target DNA of interest, hybridization, washing, and readout [89].

Pancreatic Cancer

In 2012, approximately 37,390 people in the United States died from pancreatic cancer and about 43,920 new diagnoses were made [2]. Oftentimes cancer of the pancreas develops without early warning signs and symptoms and this factors into the reason that pancreatic cancer has such a low survival rate. The survival rate for pancreatic cancer has unfortunately remained somewhat stagnant over the past 30 years with an overall 5-year-survival rate of less than 5 % [90]. The majority of people who are diagnosed with pancreatic cancer will eventually die, either from metastasis to distant organs or from local tumor invasive of the superior mesenteric artery or celiac artery, however in either case the cancer is inoperable when diagnosed at this stage [91]. Thus, when surgical intervention is appropriate the patient will have the highest chances of survival and detection and diagnostic approaches should be focused on the early stages of pancreatic cancer development. Nanobiosensors approaches offer the capability to sense small quantities of biomarkers in small sample volumes as well as improve imaging techniques and potentially advance early detection of pancreatic cancer.

Nanoparticles: There are a number of different categories of nanoparticles that have been studied in pancreatic cancer systems. SPIONs represent one of these groups that have been used in multiple experimental applications for identification and treatment of pancreatic cancer. Different targeting ligands have been utilized in successful surface functionalization for targeting of these iron oxide nanoparticles including [92]: urokinase plasminogen activator receptor (uPAR) which is a surface receptor that has increased expression on pancreatic tumor cells and stromal cells that surround the tumor [93], as well as folate. These multifunctional nanoparticles have potential for use in diagnostics, with both MR imaging and fluorescent imaging after a dye is loaded [94]. Yang et al. demonstrated that single-chained epidermal growth factor receptor antibody to the surface of either QDs or magnetic iron oxide nanoparticles results in targeted delivery to pancreatic cell and could be beneficial in drug and imaging delivery [95]. ZnO QDs have also been used as a signaling agent in sandwich immunoassays to detect CA19-9 cancer biomarkers with detection methods using both square wave stripping voltammetry and photoluminescence [96].

Another example of nanoparticles that has been used to target pancreatic cancer cells is organically modified silica nanoparticles measuring approximately 20 nm in diameter. These nanoparticles were surface functionalized with transferrin, anti-claudin 4, and antimesothelin and used the fluorophore rhodamine B as an imaging agent. This technique was found to be simple and suitable for optical bioimaging studies [97]. Similarly, mesoporous silica nanospheres where functionalized in a different study with a targeting ligand and a covalently linked Gd(III) through a disulfide moiety. It was demonstrated to be an effective MRI contrast agents in the in vitro pancreatic cancer system but when tested in vivo, the disulfide was quickly cleaved which highlighted the challenges associated with release kinetics of linker systems [98].

As mentioned earlier, there has been concern about toxicity associated with injecting humans with semiconductor metals used in construction of QDs. In response to this trepidation, over recent years, a trend has been set to engineer non-cadmium-based QD that avoid the potential barrier to widespread adoption into clinical practice. One such effort engineered QDs that were composed of an indium phosphide core and a shell made of zinc sulfide that were able to target live pancreatic cancer cells after functionalization with anticlaudin-4 or anti prostate stem cell antigen [99]. Another study took a different approach in attempting to reduce potential toxicity of CdTe/ZnS QDs by effectively encapsulating them into a triblock polymeric nanomicelle. This was effective at showing decreased toxicity while at the same time effectively accumulating in pancreatic tumors. The polymeric micelles were then conjugated with anti-mesothelin antibodies for targeting to pancreatic tumors and the size of the micelle and enclosed QD were measured to be around 120 nm [100]. Zaman et al. reported effective functionalization of QDs with a single domain antibody (2A3) which targets CEACAM6. This could be used as a new biomarker for pancreatic cancer. The authors also report that using single domain antibodies is better for targeting because they are most stable, are less likely to cause nanoparticles to aggregate, and are more cost-effective than using a traditional antibody attachment [101]. Various techniques [102] and materials have been reported that incorporate numerous targeting ligands and core materials being effective in directing QDs to pancreatic cancer for detection and diagnosis [103–107].

In addition to the other nanoparticles listed, gold and silver nanoparticles have been successfully employed for detection and diagnosis of pancreatic cancer [108, 109]. In a specific example gold nanoparticles were used to label human pancreatic cancer tissue. The authors reported that covalently attached F19 human monoclonal antibodies to gold nanoparticles were effective at labeling pancreatic carcinoma tissue and were able to visualize cancerous areas through darkfield microscopy [110]. In a different approach, functionalized gold nanorods and silver nanoparticles proved their capacity to be used as probes in pancreatic cancer detection employing darkfield microscopy or TEM [111].

Most of the experiments that use nanoparticles to view MRI images discussed so far utilize the strategy of labeling cancerous cells by targeting a receptor that has been upregulated on the surface of cancerous cells compared to normal healthy cells. An alternative approach to labeling has been proposed by a group that has created a method for labeling normal cells in pancreatic ductal adenocarcinoma. They created nanoparticles to target bombesin receptors which are known to reside on healthy cells of the pancreas. In this approach they were able to improve T2-weighted pancreatic images on magnetic resonance imaging [112].

Carbon nanotubes: Carbohydrate antigen 19-9, an important tumor antigen for pancreatic, gastric, colorectal, and hepatic cancers, has been used in the construction of a novel immunosensor incorporating carbon nanotubes, gold nanoparticles, and SiO2 nanoparticles as components. The process used to create this nanosensor was the peripheral surface of the CNT was covered with bovine serum albumin (BSA) molecules, gold nanoparticles were attached to the BSA-CNT, next electrochemical deposition of gold was added with the first gold nanoparticles providing nucleation sites for this composite layer. These initial steps provided a large surface in which CA19-9 antibody could be immobilized to and serve as a sensing component. For signal improvement and thus better sensitivity of the assay, Si02 nanoparticles were used to decorate the secondary antibody used in this sandwich immunoassay. Based upon experiments incorporating various concentrations of CA19-9 the authors demonstrated a detection limit 100 times lower than current ELISA standards used in clinical practice [113].

Multiwalled carbon nanotubes have also be used to construct a genetic fingerprint map for pancreatic cancer. In one study, the authors combined random amplified polymorphic DNA and multiwalled carbon nanotube electrochemical sensing to detect differences of guanine and deoxyguanine triphosphate in peripheral blood DNA samples of pancreatic cancer patients and controls [114].

Lung Cancer

Lung and bronchus cancer is estimated to account for nearly 226,160 new cases of cancer diagnosed in the United States. It is second among new cancer diagnosed in men and women every year behind prostate and breast cancer. However, lung and bronchus cancer account for more than three times the amount of deaths than prostate cancer and nearly twice as many deaths in women from breast cancer [2]. This makes lung cancer truly the most deadly of all cancers. The same importance of early diagnosis that has been stressed in the other cancers should be reinforced for this lethal form of cancer. Current methods of diagnosing and classifying lung cancer are imaging and biopsy-based, however these procedures are often invasive and require large imaging equipment.

An important component in lung cancer diagnosis and treatment options is differentiating various types and subtypes of lung cancer as well as stage due to the essential role this knowledge plays in selecting treatments [115]. Adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and small cell carcinoma are categories of lung tumors that have been distinguished. These categories exhibit behavior that responds differently to various treatment avenues. Thus, earlier and more accurate diagnosis of lung cancer utilizing nanobiosensor technology can play a tremendous role in the outcome of patients.

Nanoparticles: Efforts are being pursued to create valid clinical screening tests for lung cancer and other cancers based on chemical patterns recognized in exhaled breath. Barash et al. proposed a method for detection of various volatile organic compounds (VOCs) with gold nanoparticles. VOCs were collected from the headspace of numerous lung cancer cell lines. The researchers were able to create a chemical profile and determine which type and subtype of cancer cell the VOCs came from based on detection of binding gold nanoparticles (GNPs) with VOCs ligands [116]. In another study that also used a functionalized GNP-based array linked with gas chromatography–mass spectroscopy (GC–MS), it was shown that cancer patients with not only lung, but also breast, prostate, and colorectal cancers, were able to be categorized based on their breath samples [117]. While it has long been established that VOCs could differentiate lung cancer patients from healthy patients through GC–MS [118] there are drawbacks to using mass spec techniques compared to proposed nanotechnology analysis such as additional expensive equipment, time requirements of sample preparation procedures, and the need for knowledgeable technician to manage the GC–MS whereas nanobiosensors eliminate or drastically reduce some of these challenges (Table 1).

Table 1 Examples of various cancer biomarkers/targets and modalities as well as the biosensor principles that have been used for detection
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Nanowires: Similar to the discussions of other cancers mentioned, nanowires have also been used in the experimental context to capture and analyze circulating lung cancer cells. One group fabricated a quartz nanowire array and functionalized it with antibodies against epithelial cell adhesion molecules (EpCAM) which are upregulated on the surface of lung carcinoma cells. Blood samples were fed through a microfluidic cell capture apparatus and the cells were captured through binding the nanowire and then imaged with laser scanning cytometry. In this method, circulating tumor cells that only constitute a few cells per milliliter of whole blood could be detected [33].

Nanowires have also been utilized to detect two other potential biomarkers for lung cancer: IL-10 and osteopontin (OPN). A silica nanowire was used with a capture antibody that could bind either IL-10 or OPN. After binding cancer antigen to antibody, an additional detector antibody attached to an alkaline phosphatase also binds to the antigen. The second binding triggers the alkaline phosphatase to dephosphorylate the p-nitrophenyl phosphate substrate. When the reaction was measured the anodic peak current had a direct linear relationship with the concentration of the biomarkers being studied [119].

Carbon nanotubes: One example of incorporating carbon nanotubes into lung cancer detection is shown with a sensor array of single-walled carbon nanotubes that has been shown competent to distinguish breath profiles utilizing VOCs of lung cancer patients from healthy subjects [120].

Brain Cancer

Primary and metastatic brain tumors have the potential to cause devastating consequences. While brain tumors are not the most common cancer to cause death in the adult population, they represent the most common cause of cancer associated death in the pediatric population [121]. Consistent with the theme of every other cancer discussed to this point, early detection of brain cancers is associated with improved prognosis and chances of survival. Further, two challenges specific to cancers originating in the brain complicate the ability of diagnosis and treatment: (1) tight junctions of the capillary endothelium that make up the blood-brain barrier (BBB) and (2) delicate structures may be in close contact to tumor consequently instigating severe morbidity or mortality if damaged.

Nanoparticles: The brain offers a particularly promising area for nanoparticles to improve not only delivery of therapeutics but also imaging agents for better diagnosis and detection standards. The blood-brain barrier (BBB) is notable for allowing only drugs and small molecules with a molecular mass below 400–500 Daltons and high lipid solubility to cross through into the brain parenchyma [122]. These restrictions can be overcome through engineering various nanoparticles that bypass requirements of the BBB.

Fig.  1
figure 1

Cancer biomarkers can be used in the detection and diagnosis of cancer utilizing various methods of biosensing including: nanoparticles, nanowires, nanocantilevers, and nanotubes

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Many traditional imaging technologies such as positron emission computed tomography (PET), single photon emission computed tomography (SPECT), CT, X-ray, and MRI used clinically can benefit from enhancing tumor detection through nanoparticle-based modalities. The field of theranostics is working to capitalize combining diagnostic and therapeutic applications into a single preparation often utilizing current clinically available imaging systems as well as newer optical imaging. Both organic NPs as well as magnetic NPs have been used for theranostic applications in brain cancer with magnetic NPs being the most important so far due to their intrinsic MRI function [123].

One example that uses organic nanoparticles for theranostic application was shown as a proof of concept experiment using polyacrylamide nanoparticles. The NPs were functionalized with F3 peptide to target the NPs to glioma tumor vasculature. Encapsulated within the polymeric NP were iron oxide nanoparticles and a photosensitizing agent. The iron oxide is responsible for the MRI imaging element of this multifunctional nanoparticle and when the photosensitizing agent is activated by light irradiation, it generates a singlet oxygen cytotoxic component. In this system they were able to both treat and image rat gliomas in a targeted manner [124].

Further, use of functionalized magnetic NPs has been carrier out in interesting theranostic applications. In one example, a focused ultrasound was used to increase the permeability of the BBB in a locally directed manner, next magnetic targeting was applied to direct magnetic nanoparticle to the target site. This MRI monitored process led to an increased dose of cytotoxic medication that was loaded into the NP being delivered to the target [132]. Another group has taken a surface functionalization approach to magnetic nanoparticles. Glycoprotein non-metastatic melanoma protein B (GPNMB) is overexpressed by glioblastoma cells and an antibody against this target was conjugated to paclitaxel-loaded magnetic nanoparticle. This magnetic nanoparticle system was shown to effectively deliver the cytotoxic agent while being able to monitor treatment through MRI in a rat model [125]. Other examples of technologies based on magnetic nanoparticles for detection of brain cancer can be found in the literature [126, 127].

In addition, newer optical-based imaging methodologies incorporating fluorescent nanoparticles, bioluminescence, and optoacoustic tomography provide exciting possibilities. The radiation exposure from light is non-ionizing and can be used frequently without the same worry as X-ray or CT-based cumulative exposure [128]. Optical approaches have also been used to target tumors for imaging such that a near-infrared dye conjugated to a tumor-specific small peptide can show improved retention within tumor tissue being detected by a continuous-wave optical imaging system when using the rat tumor model CA20948 [129].

Nanowires: One intriguing demonstration in the application of nanowires comes from a collaboration of researchers that developed a system to use platinum nanowires with a 0.6 μm diameter to travel into the spinal cord vascular bed and record electrical stimulation. These researchers are working on improving their technique and envision replacing the platinum nanowire with a biodegradable polymer with 200 nm diameter fiber which when stimulated with an electric current could bend and be driven through the various curves intrinsic to the vascular system. Among many possibilities for monitoring the central nervous system this technology could eventually be used to help detect or monitor intracranial tumors [130].

Carbon nanotubes: Another author speculates about the potential role of carbon nanotubes and their role in intracranial monitoring. It could be possible that a carbon nanotubes could be embedded in a shunt that could then be used to detect specific sequences of DNA indicative of primary malignancy or recurrence [131].

Conclusion

This paper provides an overview of the current available nanobiosensor technologies being developed for diagnosis and detection of various cancers. The general themes discussed in the above sections focus on the need for reliable diagnostic tests with high sensitivity and specificity for early cancer detection. Nanotechnology-based platforms that meet these goals are still in their infancy but should become a valuable clinical and research tool in the coming years.