In the last chapter we present the results of our experiments. We first show a test
related to the detection of human serum albumin by using the quartz crystal analyzer,
based on monoclonal antibodies immobilized onto the sensor’s surface. We describe
the different phases of the activation of the surface, consisting of linking a thiol group
to the surface, allowing the following linking of the monoclonal antibody, and finally
we report the curves related to the addition of different concentrations of HSA (Human
Serum Albumin) in the solution in contact with the sensor.
We explain then the protocol used for the detection of DNA hybridization using the
QCM-D device. We show the structure of the synthetic oligonucleotides used, and the
protocol used for the activation of the sensor surface, consisting in the immobilization
on the gold surface of the quartz crystal of simple DNA chains of 20down-SH
oligonucleotides, by using the thiol group incorporated in one of the ends of the chains.
These chains act as recognition elements for detecting different concentrations of
100up simple DNA chains, which present a section complementary to 20down chain.
We describe the results obtained adding these complementary DNA sequences in the
solution, evaluating the frequency response of QCM-D, corresponding to the
hybridization of chains, and we finally evaluate the following recognition of the
80down DNA chain, complementary to the part of 100up that does not hybridize with
the 20down chain. We show the important results obtained, demonstrating the
possibility of detection of different concentrations of simple chains of 80 bases, and the
meaning of this contribution in the ARES project whole conception, that paves the way
to future detection of k-ras mutant sequences in a miniaturized system.
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CHAPTER I
ARES, Colorectal cancer and DNA screening
1.1 Context of the work : the ARES project
The ARES (Assembling Reconfigurable Endoluminal Surgical system) project
presents, as primary objective, the investigation and development of a prototype for a
revolutionary conception of endoluminal surgery. Endoluminal operations encompass
several distinct steps, and each one needs a dedicated technology development:
1. processing of previously acquired medical data (like images), simulation and
planning of intervention ;
2. computer design of the optimal configuration of the endoluminal robot
customized for the specific patient anatomy and for the planned therapy at the
target side ;
3. selection of modules necessary to reconstruct the robot inside the body ;
4. delivery of modules within the body to the desired site ;
5. (self) assembling and reconfiguration of the resulting kinematic chain, in order
to form the pre-planned robot ;
6. extremely precise execution of the intervention ;
7. disassembly, recovery or biodegradation of the modules.
The theoretical and technological issues are critical for implementing the concept, and
they go from the analysis of the reconfigurable internal mechanisms, to control and
communication systems development. An important and delicate phase is then the
integration of sensing and actuation modules, and it’s in this field that this work takes
place.
The specific objective on which ARES project will focus is the investigation and
development of a reconfigurable diagnostic and surgical robot for the gastrointestinal
tract; this prototype is composed by two different reconfigurable robots operating in the
gastrointestinal tract: a “screening robot”, which will search for pathologies in the GI
tract (cancer in particular) through onboard sensors, and an “interventional” surgical
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robot which will follow the screening robot to operate by microsurgical tools where
necessary.
The robotic devices will be composed of a set of heterogeneous modules (capsules)
containing different elements for structural functions, actuation, power supply,
communication, processing and control. In addition, the screening robot will host
modules with passive diagnostic devices (e.g. a camera for endoscopy and a DNA
chip), while the interventional robot will be equipped with active modules for surgery
(e.g. ablation tools, electroporation tools). The heterogeneity of modules presents some
advantages respect to a homogeneous solution: first of all we have a design tailored on
the individual module, thus reducing the number of functions per module and making
fabrication easier; moreover we can obtain a structure able to adapt its configuration to
the specific site of intervention, and to the task that must be executed (Fig. 1).
Fig. 1 : ARES configurations in different regions of the GI tract : a) ring shape for mechanical
distension of internal body wall (e.g. stomach) ; b) polyp-like shape for intervention and inchworm
locomotion in generic districts ; c) snakelike shape for negotiating narrow lumens.
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Some parts or components inside each module are common for other modules: this is
the case, for example, of the communication hardware embedding smart-tags for
mutual identification, or of module power supply, if energy is not transferred between
modules.
Each module has a volume of 0.1 – 0.5 cm
3
, and up to 10 – 15 linked modules are
supposed to be ingested at a time via a capsule in which they are stored. The capsule is
a passive carrier made out of biodegradable thin film coatings; capsules needed for the
assembly of a full robot are between 2 and 4, with individual size of about 2 cm
3
. After
ingestion, capsule film will biodegrade in the stomach, thus delivering the robot
subassemblies, that will link to each other in order to obtain the final robotic structure.
The realization of this project consists essentially in an integration of several functional
modules; some of these are directly addressed by the ARES project, and they are:
1. Electronic/Communication (EC) module : includes the master control and the
intelligence of ARES on a side, necessary to allow the self-recognition of the
initial shape and to drive the reconfiguration process, and the circuitry for
outside transmission and for possible teleoperation on the other side ;
2. Power modules : assure power supply to other modules. There are three possible
approaches to this problem:
ƒ custom power modules, that allow the use of commercial miniature
power sources, but increase the complexity of the intra-module joints,
since in order to distribute power, electrical connections are needed
between all the modules;
ƒ power sources integrated in each module, that enable the development of
intra-module joints without the special requirement of electrical
connection between the modules;
ƒ wireless energy transfer from coils located outside the patient:
electromagnetic induction will be used to provide electric power to the
receiving coils inside the robot.
3. Joint modules : allow mobility in order to change the configuration of the
ARES robot .
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There are also some modules not directly addressed by the ARES project, but that
belong to a platform on which medical micro-devices can be integrated in the robot.
They are:
9 Surgical tool module : includes mechanical tools like micro-gripper, micro-
scalpel and micro-scissors, electro-poration units, laser devices, etc.
9 Diagnostic module : carries endoscopic cameras, lab-on-chips, etc.
9 Drug delivery and storage module : may contain a drug, whose delivery is
activated as a consequence of the analysis performed by the diagnostic module.
This module can also work as a storage reservoir for biological liquids or
biopsy-tissues that will be returned to the physician for more detailed analysis.
Within diagnostic module there is the necessity to develop bio-nano systems, scaling
traditional external diagnostic tools and defining feasible modules that could equip the
screening robot. The development of diagnostic and interventional device prototypes,
therefore, is based on adaptation of existing biochemical sensors (e.g. DNA sensors)
and of existing interventional tools (e.g. electroporation tools, biopsy tools,
functionalised needles, etc.), in order to integrate them into the reconfigurable modular
structures.
In particular, the adaptation of existing nanobioanalytical sensor platforms, based on
molecular recognition, is requested. These platforms allow the detection of a wide
spectrum of antigens by the use of highly specific hybridization reactions of
complementary oligonucleotides chains. For this purpose, the antigen to detect is
specifically linked with one of the oligonucleotides chains, while the complementary
oligonucleotide chain remains immobilised on the detection platform. This platform
could be used for the detection of different types of antigens, and consequently it can
feature a “universal” detection. To fix the oligonucleotides to the surfaces, a novel
functional nanostructured polymers can be used. Usually, the detection is made by
using fluorescence markers linked to the antibodies specific to the antigens to be
detected, but this technique requires the integration of optical detectors. An important
aim of the project is to analyze the capabilities of an electrical detection platform; the
oligonucleotides will be immobilised on the gap of nanoelectrodes, by means of novel
functionalization techniques: this platform will avoid the use of fluorescence markers.
The detection will be performed through the measurement of the change in the
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electrical properties, when the hybridization of the oligonucleotides takes place. The
level of signal may be incremented by using specific antibodies.
The integration in ARES of a nanobioanalytical sensor capable of DNA analysis,
therefore, would represent an outstanding result in order to achieve the capability of
early cancer screening in the colorectal tract.
1.2 Analysis of the pathology: the Colorectal Cancer
In order to obtain an efficient diagnostic instrument, the study of cancer development
and of mechanism on which is based its capability to avoid apoptosis and other body’s
defence systems, assumes a primary importance. This type of analysis, in fact, allows to
understand and to find which are the targets of the diagnosis and of an eventual
therapy, and above all which are the molecular changes involved, in order to create
systems capable to detect them with a high specificity.
Colorectal cancer includes cancerous growths in the colon, rectum and appendix. We
are mainly interested in tumour formations at colon level, commonly indicated as colon
cancer. This is the third most common form of cancer and the second leading cause of
death among cancers in the Western world.
1.2.1 Anatomy and function of the digestive system
In the anatomy of the digestive system, the colon (from the Greek word: κῶλον) is the
part of the large intestine between the cecum to the rectum [1-2]. Its primary purpose is
to extract water from feces. In mammals, it consists of the ascending colon, transverse
colon, the descending colon, and the sigmoid colon. The colon from cecum to the mid-
transverse colon is also known as the right colon. The remainder is known as the left
colon. The location the colon parts are either in the abdominal cavity or behind it in the
retroperitoneum. The colon in those areas is fixed in location.
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Fig. 2 : Anatomic description of Large Intestine
The ascending colon, on the right side of the abdomen, is about 12.5 cm long. It is the
part of the colon from the cecum to the hepatic flexure (the turn of the colon by the
liver). It is retroperitoneal in most humans. In grazing animals the cecum empties into
the spiral colon. Anteriorly it is related to the coils of small intestine, the right edge of
the greater omentum, and the anterior abdominal wall. Posteriorly, it is related to the
iliacus, the iliolumbar ligament, the quadratus lumborum, the transverse abdominis, the
diaphragm at the tip of the last rib; the lateral cutaneous, ilioinguinal, and
iliohypogastric nerves; the iliac branches of the iliolumbar vessels, the fourth lumbar
artery, and the right kidney. The transverse colon is the part of the colon from the
hepatic flexure (the turn of the colon by the liver) to the splenic flexure (the turn of the
colon by the spleen). The transverse colon hangs off the stomach, attached to it by a
wide band of tissue called the greater omentum. On the posterior side, the transverse
colon is connected to the posterior abdominal wall by a mesentery known as the
transverse mesocolon. The transverse colon is encased in peritoneum, and is therefore
mobile (unlike the parts of the colon immediately before and after it). More cancers
form as the large intestine goes along and the contents become more solid (water is
removed) in order to form feces. It is primarily supplied by the middle colic artery, a
branch of superior mesenteric artery. The descending colon is the part of the colon from
the splenic flexure to the beginning of the sigmoid colon. It is retroperitoneal in two-
thirds of humans, and in the other third it has a (usually short) mesentery. The sigmoid
colon is the part of the large intestine after the descending colon and before the rectum.
The name sigmoid means S-shaped. The walls of the sigmoid colon are muscular, and
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contract to increase the pressure inside the colon, causing the stool to move into the
rectum. Due to the intermittent high pressure within it, the colon can develop pockets
called diverticuli in its walls. The presence of diverticuli, whether harmful or not, is
called diverticulosis. An infection of the diverticuli is called diverticulitis.
For what concerning the function [3], the large intestine comes after the small intestine
in the digestive tract and measures approximately 1.5 meters in length. Although there
are differences in the large intestine between different organisms, the large intestine is
mainly responsible for storing waste, reclaiming water, maintaining the water balance,
and absorbing some vitamins, such as vitamin K. By the time the chyme has reached
this tube, almost all nutrients and 90% of the water have been absorbed by the body. At
this point some electrolytes like sodium, magnesium, and chloride are left as well as
indigestible carbohydrates known as dietary fiber. As the chyme moves through the
large intestine, most of the remaining water is removed, while the chyme is mixed with
mucus and bacteria known as gut flora, and becomes feces. The bacteria break down
some of the fiber for their own nourishment and create acetate, propionate, and butyrate
as waste products, which in turn are used by the cell lining of the colon for
nourishment. This is an example of a symbiotic relationship and provides about one
hundred Calories a day to the body. The large intestine produces no digestive enzymes :
chemical digestion is completed in the small intestine before the chyme reaches the
large intestine. The pH in the colon varies between 5.5 and 7 (slightly acidic to neutral).
1.2.2 Characteristics, symptomatology and ethiopathology
Many colorectal cancers are thought to arise from adenomatous polyps in the colon
(Fig. 3). These mushroom-like growths are usually benign, but some may develop into
cancer over time.
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Fig. 3 : Polyp of sigmoid colon as revealed by colonoscopy.
Approximately 1 cm in diameter.
A polyp is an abnormal growth of tissue (tumor) projecting from a mucous membrane.
If it is attached to the surface by a narrow elongated stalk it is said to be pedunculated
and if no stalk is present it is said to be sessile. Polyps are commonly found in the
colon, stomach, nose, urinary bladder and uterus. They may also occur elsewhere in the
body where mucous membranes exist like the cervix and small intestine. Colon polyps
are uncommonly associated with symptoms. Occasionally rectal bleeding, and on rare
occasions pain, diarrhea or constipation may occur because of colon polyps. Colon
polyps are a concern because of the potential for colon cancer being present
microscopically and the risk of benign colon polyps transforming with time into colon
cancer. The majority of the time, the diagnosis of localized colon cancer is through
colonoscopy. Therapy is usually through surgery, which in many cases is followed by
chemotherapy.
Frequently, the patient may be asymptomatic. This is one reason why many
organizations recommend periodic screening for the disease with fecal occult blood
testing and colonoscopy, and this is the reason why there is the necessity of the
development of an innovative early screening system for this pathology, like the DNA
biosensor that we want to create. When symptoms do occur, they depend on the site of
the lesion. Generally speaking, the nearer the lesion is to the anus, the more bowel
symptoms there will be, such as:
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