can be used as targets in their detection, isolation and quantification. In consequence, cancer cells could in theory be detected by the electrocatalytic sensing techniques developed for the sensing of proteins.
Figure 4. Schematic representation of the antigens expression at the surface of cancer cells.
Numerous optical detection methods were developed using nanomaterial optical probes for the detection of various cancer cell models, but few works describe the use of nanoparticle labels for their electrochemical detection.
Several groups based their electrochemical cytosensors on the recognition of surface carbohydrates and glycopeptides, and incorporated nanomaterials to enhance the detection. Din et al. developed a label-free strategy for facile electrochemical analysis of dynamic glycan expression on living cells. They used carbon nanohorns to efficiently immobilize lectin for the construction of a recognition interface and enhancing the accessibility of cell surface glycan motifs. [49] Other electrochemical cytosensor was designed based on the specific recognition of mannosyl on a cell surface to concanavalin A (ConA) and the signal amplification of gold nanoparticles (AuNPs). By sandwiching a cancer cell between a gold electrode modified with ConA and the AuNPs/ConA loaded with 6-ferrocenylhexanethiol (Fc), the electrochemical cytosensor was established. The cell number and the amount of cell surface mannose moieties were quantified by cyclic voltammetry (CV) analysis of the Fc loaded on the surface of the AuNPs. Since a single AuNP could carry
Chapter1. General introduction
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hundreds of Fc, a significant amplification for the detection of target cell was obtained. They used K562 leukemic cells as model, and observed that the electrochemical response was proportional to the cell concentration in the range from 1.0×102 to 1.0×107 cells mL-1 showing very high sensitivity. [50]
Figure 5. Schematic representation of the reusable aptamer/graphene-based aptasensor. The sensor is constructed based on graphene-modified electrode and the first clinical trials II used aptamer, AS1411. AS1411 and its complementary DNA are used as a nanoscale anchorage substrate to capture/release cells.
Feng et al. reported an electrochemical label-free sensor for cancer cell detection using the first clinical trial II used aptamer AS1411 and functionalized grapheme (Fig. 5). By taking advantages of the aptamer high binding affinity and specificity to the overexpressed nucleolin on the cancer cell surface, the developed electrochemical aptasensor could distinguish cancer cells and normal ones and detect as low as one thousand cells. This sensor could also be regenerated and reused. This work is a good example of label-free cancer cell detection based on aptamer and graphene-modified electrode. The authors claim that this graphene/aptamer-based design can be adaptable for detection of protein, small molecules, and nucleic acid targets by using different aptamer’s DNA sequences. [51]
Other groups based their cytosensors on the recognition of membrane protein receptor supposed to be specific for the particular cancer type under investigation.
Li et al. proposed a sensitive electrochemical immunoassay to detect breast cancer cells by simultaneously measuring two co-expressing tumor markers, human mucin-1 and carcinoembryonic antigen (CEA) on the surface of the
! 18!
revealed that a proper electrochemical response could only be observed under the condition that both of the tumor markers were identified on the surface of the tumor cells. With this method, breast cancer cell MCF-7 could be easily distinguished from other kinds of cells, such as acute leukemia cells CCRF-CEM and normal cells islet beta cells. Moreover, the prepared cytosensor could specially monitor breast cancer cell MCF-7 in a wide range from 104 to 107 cell mL-1 with fine reproduction and low detection limit, which may have great potential in clinical applications.[52]
Other group [53] reported a single nanotube field effect transistor array, functionalized with IGF1R-specific and Her2-specific antibodies, which exhibits highly sensitive and selective sensing of live, intact MCF7 and BT474 human breast cancer cells in human blood. Single or small bundle of nanotube devices that were functionalized with IGF1R-specific or Her2-specific antibodies showed 60% decreases in conductivity upon interaction with BT474 or MCF7 breast cancer cells in two µl drops of blood. Control experiments produced a less than 5% decrease in electrical conductivity, illustrating the high sensitivity for whole cell binding by these single nanotube-antibody devices. They suggest that the free energy change, due to multiple simultaneous cell-antibody binding events, exerted stress along the nanotube surface, decreasing its electrical conductivity due to an increase in band gap.
They reported this achievement as a nanoscale oncometer with single cell sensitivity with a diameter 1000 times smaller than a cancer cell that functions in a drop of fresh blood.
Circulating Tumor Cells (CTCs) are blood-travelling cells that detach from a main tumor or from metastasis. CTCs quantification is under intensive research for examining cancer metastasis, predicting patient prognosis, and monitoring the therapeutic outcomes of cancer. [1–5] Although extremely rare, CTCs detection/quantification in physiological fluids represents a potential alternative to the actual invasive biopsies and subsequent proteomic and functional genetic analysis.[6,7]
Therefore their discrimination from normal blood cells offers a high potential in tumor diagnosis.[4,8] Established techniques for CTC identification include labeling cells with antibodies (immunocytometry) or detecting the expression of tumor markers by reverse-transcriptase polymerase chain reaction (RT-PCR).[9] Recently, several works that employ nanomaterials for optical detection of CTCs were reported.
Chapter1. General introduction
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Conclusion and future perspectives
The application of NPs as catalysts in biomarker detection systems is related to the decrease of overpotentials of many important redox species including also the catalyzed reduction of other metallic ions used in labeling-based protein sensing.
Although the most exploited materials in catalysis are the metals from platinum group, with the introduction of nanotechnology and the increasing interest for biosensing applications, gold NPs, due to their facile conjugation with biological molecules, besides other advantages, seem to be the most used metal nanoparticles. Their applications as either electrocatalytic labels or modifiers of protein related transducers are bringing important advantages in terms of sensitivity and detection limits in addition to other advantages.
The employment of carbon based nanomaterials like grapheme oxide underwent an intensive progress in the last few years. Their ability to electrocatalyze different electrochemical reactions together with a better control on the synthesis and its electrochemical properties, promises an exponential growth of new reported applications in the biosensing field.
Cancer cell detection could also profit from the use of electrocatalytic nanomaterials, to improve the current limitations in terms of sensitivity and simplicity of the assay. Since the cancer cells express specific proteins or carbohydrates at their plasma membrane, in theory the electrocatalytic properties of NPs used in protein-biomarker detection could also be extended to cell analysis. Interesting new strategies for the electrochemical detection of cancer cells, could be much appreciated due to the excellent characteristics of these biosensing techniques that include fast and sensitive responses, easy to use equipment, handling of low volume of samples, portability and the possibility to be integrated in fluidic devices, so as to achieve point-of-care detection instruments.
For this reason, the cancer detection is one of the objects of study in the following chapters of this thesis. For example the hydrogen catalysis reaction induced by AuNPs applied for protein detection (chapter 3) was also studied for cancer cells detection. A new strategy that comprises the capturing of CTCs, from among other cells, and includes also their fast labeling/detection, by means of fast and easy-to-use electrochemical systems, is supposed to be ideal for point-of-care diagnostics and therefore was also developed (chapter4).
So as to conclude, the conjugation of NPs with electrochemical sensing systems promises large evolution in actual electroanalysis methods and is
! 20!
based sensing systems have shown high sensitivity and selectivity, their implementation in clinical analysis still needs a rigorous testing and control period so as to really evaluate these advantages in comparison to classical assays in terms of reproducibility, stability and cost while being applied for real sample analysis. Further developments including the development of simple electrochemical devices (i.e. pocket size such as glucosimeter) or fluidic integrated devices are necessary for future entrance in real sample diagnostics in terms of point-of-care biosensors.
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8M This thesis is divided in 7 chapters. A brief explanation of each chapter is giving in the following part.
Chapter 1, titled “General Introduction”, is an introductory chapter that comprises two parts. The first is the thesis overview that briefly describes each of the seven chapters in which the thesis manuscript is divided. The second part is a review on the state-of-the art of the sensing technologies based on electrocatalytic nanomaterials. The review focuses the application of nanomaterials for biomarker electrochemical detection in general, with more detail on protein and cancer cell detection. The DNA detection is not addressed in this chapter, even though DNA sequences can be considered biomarkers when a genetic disorder is implicit. Nevertheless a book chapter, written owing to the book editor invitation, is discussed in chapter 5.
(The introductory review is in preparation for its subsequent submission).
Chapter 2, “Objectives”, introduces the objectives that motivated and guided this work.
Chapter 3, “Electrocatalytic nanoparticles for protein detection”, describes the development of two electrocatalytic methods, based on gold nanoparticles.
The first one is the electrocatalytic deposition of silver onto AuNPs and the second one the electrocatalyzed hydrogen evolution reaction (HER). The application of both methods on electrochemical immunoassays for HIgG detection (as a model protein), the application of the second for the quantificatiozn of Hepatitis-B antibodies in real human samples are also described.
Chapter 4, “Electrocatalytic gold nanoparticles for cell detection”, describes the application of AuNP based HER, explained in the preceding chapter, to the detection of cancer cells using different approaches. The first approach shows the detection of adhered cancer cells and the second one the detection of circulating tumor cells
Chapter 5 , “Additional works”, comprises two parts. The first part briefly describes the state-of-the-art of the nanoparticle-induced catalysis for electrochemical DNA biosensors. The second describes an additional work
! 25!
concerning the application of CNTs composites for the electrochemical detection of thrombin spiked in human serum.
Finally, in Chapter 6, “Conclusions and future perspectives”, the general conclusions are presented, as also as the future perspectives on the application of the work described in the previous chapters. In Chapter 7, are displayed the publications and manuscripts that resulted from this thesis research (as explained in the Preface).
Chapter 2
Objectives
8A
Chapter 2
Objectives The main objective of this thesis is to develop novel and improved electrochemical sensing systems for biomarker detection, exploiting the electrocatalytic effects of nanomaterials in general and nanoparticles particularly.
Detailed objectives