1.
INTRODUCTION
1.1
Biosensor Definition and Fields of Use
In recent years, the use of biosensors has been recognized as having potential
in widespread analytical applications. A biosensor can be defined as “a self-contained
integrated device that is capable of providing specific quantitative or semi-quantitative
analytical information using a biological recognition element (biochemical receptor)
which is in direct spatial contact with a transduction element.[1]” A bioanalytical
sensor incorporates a biological element such as an enzyme, antibody, nucleic acid,
microorganism or cell. Currently, bioanalytical sensors have been utilized in three
main areas[2]:

Medicine and healthcare for clinical diagnosis, pharmaceutical and drug
analysis, and bacterial and viral analysis.

Environment for pollution control and monitoring

Quality assurance and process control for fermentation, food and drink
Biosensors have many advantages over other detection methods. Use of
biologically derived compounds allows specific analyte measurements with great
accuracy. Unlike other bioanalytical methods such as ELISA and other immunoassays,
the analyte can be measured directly and instantaneously; there is no need to wait for
results from lengthy procedures that must be carried out in laboratories. By
combining the receptor and transducer into one single sensor, a biosensor enables
measurement of target analytes without requiring steps that uses many reagents to treat
the sample. Thus, the detection method is greatly simplified. Another benefit of
1
biosensors over traditional detection methods such as immunoassays and ELISA is the
highly selective, fast, simple, and continuous detection that occurs with biosensors. In
fact, biosensors promise several distinct advantages to the health-care sector over
conventional analytical techniques. Biosensor-based medical instruments can be used
for the monitoring of patients during intensive care and surgery and diagnosing
patients. Biosensors are attractive for following reasons [3,4,5]:

They may be designed as inexpensive, disposable devices requiring little or no
expertise for their use.

They allow the quantitative detection of specific species in complex samples
without the need for sample processing or purification.

These features provide the opportunity for sample analysis to be performed
outside hospital’s analytical laboratory to the doctor’s office and the patient’s
bedside, and for self-testing in the home.

The volume of sample required can be reduced from ml to μl.

Results may be obtained immediately in situations where delay may be critical,
for example in ICU Departments, or sensors may be implanted to allow
continuous monitoring of patient status

Multi-analytical devices allow quantification of several species simultaneously.
One area of heath care sector that can utilize biosensors is in cell enumeration using
immunophenotyping. The immunophenotyping of cell is commonly used in clinical
medicine as a way to assess the patient’s condition and monitor the disease
progression.
2
1.2
Immunophenotyping of Human Blood Cells
The human immune system is comprised of organs and leukocytes that defend
the body against disease and infection. The leukocyte family consists of
polymorphonuclear neutrophils, lymphocytes, monocytes, and basophils, which
function uniquely from each other. Leukocyte cell surface molecules are named
systematically by assigning them a cluster of differentiation (CD) antigen number that
includes any antibody that has an identical and unique reactivity pattern with different
leukocyte populations. Table 1 lists some CD antigens and the cells that express them.
CD antigen
Cellular expression
CD2
T cells, thymocytes, NK cells
CD3
Thymocytes, T cells
CD4
Thymocyte subsets (helper & inflammatory T cells), monocytes,
macrophages
CD8
Thymocyte subsets (cytotoxic T cells)
CD15
neutrophils, eosinophils, monocytes
CD16
neutrophils, NK cells, macrophages
CD18
Leukocytes
CD19
B cells
CD28
T cell subsets, activated B cells
CD29
Leukocytes
CD30
Activated B and T cells
CD34
hematopoietic precursors, capillary endothelium
CD36
platelets, monocytes
CD40
B cells, monocytes, dendritic cells
CD45
Leukocytes
CD46
hematopoietic and non-hematopoietic nucleated cells
CD48
Leukocytes
CD72
B cells
Table 1: List of some CD antigens specific for cell types. [6]
3
Different subtypes of leukocytes can be distinguished from using the
combinations of receptor proteins on the surface of the cell. For example, all
leukocytes contain CD45 receptors on their surface. Lymphocytes circulate in the
blood and aid in defending the body against infections and the intrusion of foreign
materials. Two main classes of lymphocytes are T cells (produced from the thymus)
and B cells (produced from the bone marrow). All T cells have CD3 protein on the
surface membrane. Current cell enumeration techniques have used the surface
markers for separation and identification.
1.3
Cell Enumeration Applications
1.3.1
Cell Enumeration for Human Immunodeficiency Virus Diagnostics
1.3.1.1 HIV Background
The human immunodeficiency virus (HIV) infection can lead to Acquired
Immunodeficiency Syndrome (AIDS) and ultimately to death. According to the Joint
United Nations Program on HIV/AIDS (UNAIDS), approximately 37.8 million people
live with HIV, and 20 million people have died from AIDS as of the year 2003 [7].
Early HIV detection allows early application of antiretroviral therapy that can prevent
AIDS and prolong health. The antiretroviral therapy works by reducing the amount of
virus in the body and can dramatically slow the deterioration of the immune system.
For example, the government of Brazil estimates that antiretroviral treatments, that
suppress activities of retrovirus such as HIV, have decreased mortality rates by 50%,
morbidity rate by 60-80%, and the hospitalization of HIV positive people by 70%.[7]
4
1.3.1.2 HIV Diagnosis
HIV binds to CD4 receptors on the T helper cells (CD4 cells) and then uses the
lymphocyte as a host to multiply its genes. Thus with the increase in the number of
HIV in the blood, more CD4 cells get infected and destroyed which leads to a
significant drop in CD4 cells in the patient’s blood. In the United States, two types of
techniques are used to detect HIV infections in people. One technique directly detects
the HIV-RNA concentration in blood samples and the other one indirectly detects HIV
level by determining the number of CD4 cells in the patient’s blood. The CD4
absolute count requires the total white blood cell count, the percentage of white cells
that are lymphocytes, and the percentage of lymphocytes that express CD4. [8]
An HIV infection can cause AIDS principally because of the depletion of CD4
lymphocytes. Healthy people have approximately 435 to 1500 CD4 cells per micro
liter of blood [9]. Once a person is infected with HIV, the virus target CD4 cells and
replicates at an exponential rate while destroying the CD4 cells. Then the immune
system reaches a point where the rate of viral replication exceeds the rate of CD4 cell
production. As a result, the immunologic function of the body weakens, and the body
becomes more susceptible to diseases and other viral infections. Antiretroviral drugs
can reduce the concentration of virus and thus stop further reduction of CD4 cells [8].
Antiretroviral therapies are initiated when the CD4 cell count is less than 350 cells per
micro liter [9] or 14% of total number of lymphocytes [10].
CD4 cells bear CD3, CD4, and CD 19 receptors on the surface as well as
CD 45 receptors. These receptors exist in other cell types as well, but only T helper
cells have a combination of all CD3, CD4 and CD45. This characteristic of T helper
5
cells is used in the determination of the concentration of CD4 cells by flow cytometry,
since the progressive depletion of CD4 cells is the chief event in pathogenesis during
an infection by HIV. The absolute number of these CD4 Cells in the peripheral blood
is the single most important parameter for monitoring the disease associated with HIV
infection. Thus, CD4 cell enumeration is essential in three areas.
First, initial CD4 counts are used to find the degree of a person’s immune
deterioration. The CD4 tests are repeated to monitor a change in the CD4 count and to
define a declining slope of CD4 counts as an indication of the speed of progression
toward AIDS. With the help of baseline CD4 counts, patients are placed in care to
start antiretroviral therapy and to define a starting point for “efficient prophylaxis
against opportunistic infections.” Second, while the patient is on the antiretroviral
therapy, improvements in CD4 counts indicate whether the therapy is effective.
Finally, CD4 counts are used to prepare for an anticipated health care need in any one
region through an epidemiological AIDS surveillance[11]. Thus, flow cytometry
provides a methodology for multiparameter analysis of blood cells, at the single cell
level.
The analyte of major interest is the absolute CD4 count, however that number
is meaningless by itself, as opposed to that the result in the context of a full complete
blood count (CBC). Therefore, in addition to the absolute CD4, the CD4 percentage,
along with CD3 percentage and CD3 absolute count are also reported. In fact, this
lymphocyte subset analysis, entailing determination of percentages and absolute
counts for CD3 (pan T), CD3/4 (T-helper), CD3/8 (T-cytotoxic/suppressor), CD16+56
(NK) and CD19 (pan-B) is performed on all submissions from patients with any
6
suspected immunodeficiency condition. Though the latter three are not reported for
routine CD4 requisitions, the data is available if needed. The additional analyses are
performed to facilitate internal quality control for the test.[12]
1.3.2
Other Cell Enumeration Applications
Cell enumeration can be used for lymphocyte subset analysis for diagnosis and
evaluation of the immunological status of a patient. Changes in the lymphocyte
subsets can indicate immunological changes produced by various diseases or
treatments. Helper/suppressor cell counts, including total T cell (CD3), helper T cell
(CD4), suppressor cell (CD8), natural killer cells (CD16, CD56), and white blood cell
and absolute lymphocyte counts, are used as a general screening in cases where there
is no knowledge of the underlying cause of disease.[13]
Lymphocyte subsets (CD3 and CD4 T cells and CD19 B cells) are monitored
on patients undergoing bone marrow transplants with peripheral stem cell products
and chemotherapy. Cell numbers can be used in evaluating these patients for disease
remission, or transplant rejection. After an organ transplant, CD3 cell level is
measured during immunosuppressive therapy. This therapy causes a disappearance of
T cells. Thus, steady rise in the T cell demonstrates a failure to respond to therapy.
Immunophenotyping of CD34 is used to determine the number of
hematopoietic stem and progenitor cells. These stem cells are necessary for
reconstituting the bone marrow. Bone marrow transplant patients undergo a series of
procedures that enhance CD34 production, collect CD34-enriched products, destroy
the diseased bone marrow and then return the CD34 enriched products back to the
7
patient.[14] More extensive cell enumeration by immunophenotyping is used to
distinguish Chronic Lymphocytic Leukemia from a generalized increase of
lymphocytes or to distinguish Acute Lymphoid Leukemia from Acute Myeloid
Leukemia. [15]
1.4
Cell Enumeration Methods
1.4.1
Flow Cytometric Immunophenotyping
Flow cytometric immunophenotyping is the golden standard for cell
enumeration and has been used routinely in diagnostic analyses since its first
commercialization in the 1950s.[16] Flow cytometry uses a cytometer to
simultaneously and rapidly measure various physical and chemical properties of single
particles or cells as they “flow in a fluid stream one by one” through a beam of
light[17]. Each cell is correlated with its own characteristic scattered lights and
fluorescent lights[18].
A flow cytometer consists of three main systems: a fluidic system, an optics
system, and an electronics system. The fluidic system contains a flow chamber that
transports particles or cells in suspension and sheath fluid in a single file. The
transported particles then pass by the optics system, which has a light source (e.g.,
laser), filters, and a light collecting lens. Then the detector converts the collected light
signals into electronic signals that can be processed and displayed as a graph on the
computer screen. For cell enumeration, fluorescence detection is used in addition to
side and forward scatter detection. A fluorescent probe is utilized to label other probes
through covalent bonding and to indicate particular composition of the cell or to
8
directly stain the cell to analyze cell structure[19]. In the case of cell immunotyping,
fluorescent probes are coupled to anti-antibodies, which bind to specific surface
receptors, and distinguish sub-populations of differentiated cell types.[20]
Initially, a dual-platform (two instruments) technology has been used for cell
enumeration. For the measurements using dual-platform technology, the absolute
lymphocyte count generated from the hematology analyzer is combined with cell
count, for example CD4 T cells, from the flow cytometer to obtain a CD4 percentage
of lymphocytes.[21] Using two instruments, however, increases human handling of
samples and decreases accuracy. Difficulty arises in direct comparisons of
hematology results between different types of instruments in different laboratories
because of the time constraint in shipping blood to multiple locations.[22]
Demand for accurate cell counts led to a development of a single-platform
technology where the absolute numbers of lymphocyte populations as well as CD cells
are obtained only by flow cytometry [23]. Single platform analysis can be done by
using different color fluorochromes. Adding more fluorochromes to the analysis
allows the gathering of more information, uses less sample volumes and thus results in
a lower number of tubes, and requires less preparation and analysis time per
patient.[19]
One disadvantage of flow cytometer is the requirement of specialized
equipment, such as the Beckton Dickinson FACS-Calibur or Coulter Systems
cytometers. The equipment is quite expensive (at least $50,000) and each assay bears
significant costs, for example the fluorochrome-conjugated antibodies can cost $20 to
$30 per test [24].
9
1.4.2. CD4 Enumeration Techniques
In the case of CD4 enumeration, other diagnostic strategies that minimize the
amount of specialized equipment used are available. For example, the Coulter Manual
CD4 Count kit allows manual count of CD4 cells using microscopy by employing
latex spheres coated with murine monoclonal antibody. Crystal violet–stained CD4+
T lymphocytes with beads attached are identified by counting in a hemocytometer.[25]
The Dynabeads T4-T8 system is very similar, to the Coulter Manual CD4
Count kit. It uses antibody coated magnetic particles (Dynabeads). Here, the CD4
cells captured by the dynabeads are separated from the whole blood by a magnet, the
cells are lysed and the nuclei are stained with Sternheimer-Malbin staining solution
(containing crystal violet, safranin O, ammonium oxalate, and ethanol and counted.
Both systems also use beads coated with another antibody, anti-CD14, to remove
monocytes from CD4 T cells.[25]
1.5
Objective
The objective of this project is to design a biosensor that quantifies the amount
of T-lymphocytes in human blood. A microfluidic biosensor based on liposome signal
generation and amplification is adapted to the detection of the T-lymphocytes using
two specific sets of antibodies recognizing proteins on the surface of the cells. The
project focused on the design and optimization of the device in order to produce an
accurate, reliable detection system.
10
2.
DESIGN
2.1
Analyte
The analytes of interest are T lymphocytes in human blood. A sandwich
detection approach is used in order to separate all T-lymphocytes from the remaining
cells in the blood sample. Thus, the CD45 marker is used to separate the leukocytes
from other cells in the blood sample. CD3 maker is used to separate the T
lymphocytes for subsets of leukocytes. Overall, CD3 T cell counts can be used as
positive control in CD4 enumeration for HIV detection and for monitoring
immunosuppressive therapy.
2.2
Liposome-Bead Sandwich Detection
Liposomes are small, spherical vesicles that spontaneously form when
phospholipids molecules in aqueous solution self-assemble into hollow spheres with
an aqueous cavity enclosed by a phospholipids bilayer membrane. Fluorescent dyes
can be encapsulated in the liposomes for bioanalytical assay applications as has been
shown in numerous applications previously [27,28,29]. Dye encapsulated liposomes
are advantageous as signal generators because encapsulation of many dye molecules in
one liposome allow for signal amplification leading to extremely low detection limits..
The detection method developed utilizes immunoliposomes and
superparamagnetic beads that use biotin-streptavidin interactions to immobilize antiantibodies. Biotinylated anti CD45 antibodies are immobilized onto supermagnetic
beads coated with streptavidin and biotinylated anti CD3 antibodies are immobilized
11
onto streptavidin-tagged liposomes. The immuno-liposomes, immuno-beads, and
blood sample are incubated. During incubation, the anti-antibodies bind to the
specific proteins present on the lymphocytes and produce a sandwich formation as
seen in Figure 1.
Figure 1: Diagram of trapped of immunobeads in the capture zone of the microfluidic device.
Some immunobeads are bound to the white blood cells and subset of those for a sandwich
complex as the immunoliposome bind to the T-lymphocytes.
This sandwich structure is isolated from the rest of the blood sample by using
a magnetic collection of the beads to the rare-earth neodymium-iron-boron magnet
placed in a microfluidic channel (see chapter 3.5). Fluorescence from entrapped dye
can be detected using a fluorescence microscope.
2.3
Microfluidic Device
Use of microfluidics in analytical systems has increased due to its high
throughput capacity and smaller sample volume requirement [30]. A microfluidic
device designed previously in this laboratory has been applied to the analysis of T-
12
lymphocytes in this project. The microfluidic layout is shown in Figure 2 and will be
discussed in more detail in chapter 3.5.
PDMS film
Glass slide
Plexiglas plate
screws
Figure 2: Top view of assembled microfluidic sensor system with two Plexiglas housing the
PDMS with channel grooves and the class slide. 8 screws are used to hold the Plexiglas plates
together
The biosensor is made in polydimethyl siloxane (PDMS) and packed in
Plexiglas to provide connection to the outside world. The well made in the Plexiglas
on top of the detection zone provides a place for the magnet. The externally placed
magnet allows a way of controlling magnetic beads within the microchannel. This
system allows less sample size, human handling, and time. Mixture of liposomes with
reporter probes (anti CD3 antibodies), beads with capture probes (anti CD45
antibodies), and target (blood sample) are incubated prior to introduction into the
microchannel. The sandwich complexes are captured on the magnet and detected by
fluorescence microscope.
13
3.
MATERIALS AND METHODS
3.1
Materials and Reagents
All general laboratory chemicals and buffer reagents were purchased from
Sigma Chemical Company, St. Louis, MO. Organic solvents were purchased from
Aldrich Chemical Company, Milwaukee, WI. Lipids were obtained from Avanti Polar
Lipids, Alabaster, AL. Sulforhodamine B and Streptavidin were purchased from
Molecular Probes Company, Eugene, OR. Superparamagnetic beads (Dynabeads
MyOne Streptavidin) were purchased from Dynal Biotech Inc., Lake Success, NY.
Silicone elastomer kit Sylgard-184 containing Polydimethyl Siloxane (PDMS)
prepolymer and catalyst was obtained from Dow Corning Corp., Midland, MI. The
Cornell Nanofabrication Facility (CNF) provided clean room facilities, chemicals and
equipment for silicon template fabrication. The Plexiglas housing was constructed in
the machine shop located at the School of Chemical and Biomolecular Engineering,
Cornell University. Anti-CD3 and CD45 antibodies were purchased from Biodesign
International, Saco, Maine.
3.2
Immunoliposome Preparation
3.2.1
Liposome Preparation
Liposomes were prepared using the reversed-phase evaporation method.
Initially, the encapsulant was prepared by dissolving 150mM Sulforhodamine B
(SRB) in a 0.2M HEPES with pH 7.5 and DIUF water. A vapor pressure osmometer
was used to measure the osmolarity of the encapsulant. Osmolarity of the encapsulant
14
in 0.2M HEPES was 630 mmol/kg and the osmolarity of the buffer, 0.01M HEPES
pH 7.5 was 365mmol/kg. 304.9g of sucrose was added to the buffer to adjust the
osmolarity to be 75 mmol/kg greater than the encapsulant. 7.2μmol of diphosphatidyl
palmitoylethanolamine (DPPE) was then dissolved in 2mL of 0.7% triethylamine (v/v)
in chloroform by sonicating for one minute at 45°C in a round-bottom boiling flask.
Then 14.3μmol of N-succinimidyl-S-acetylthioacetate (SATA) was added to the
DPPE suspension, sonicated again, and placed on a shaker for 20 minutes, forming the
DPPE-ATA compound. The triethylamine was removed by adding 3 mL of
chloroform to the mixture and evaporating it under a vacuum in a rotary evaporator at
45°C.
The lipids 40.03μmol dipalmitoyl phosphatidylcholine (DPPC), 21.0μmol
dipalmitoyl phosphatidylglycerol (DPPG), 51.7μmol cholesterol and the 30.6μmol
DPPE-ATA were dissolved in a mixture of 3mL chloroform, 0.5ml methanol and 3mL
isopropyl ether. The lipid and organic solution mixture was sonicated in 45°C bath for
30seconds. Then 4mL of SRB encapsulant was added to the organic solution and
sonicated for 4 additional minutes until the lipids were completely dissolved. Vacuum
rotary evaporator was used to evaporate the excess liquid at the highest rotation speed
with the vacuum 380 mbar/hPa for 30 minutes then with the vacuum 280 mbar/hPa for
additional 30 minutes. The flask with thick viscous organic solvent was vortexed.
Then 4mL of SRB encapsulant was added to the lipid mixture and sonicated for five
minutes. The lipids spontaneously formed liposomes entrapping the SRB dye.
To obtain uniform liposome size, they were extruded through 2 μm (11 times)
and then 0.6μm (11 times) polycarbonate filters from Avanti Polar Lipids using the
15
Avanti mini extruder. The extrusion process was completed at a temperature between
50 to 60°C. These liposomes were purified by gel filtration using a Sephadex G50
column equilibrated with 0.01M HEPES, 0.2M sucrose, pH7.5 solution. After the
liposomes were collected from the column, a measurement of the optical density of the
liposomes was taken using the spectrophotometer. The spectrophotometer was
blanked using the 0.01M HEPES buffer solution. 10μL of liposomes were diluted in
990μL of HEPES buffer. A plot of absorbance at 532 nm versus sample number
allowed the high and medium fractions of the liposomes to be determined (Figure 3).
At this wavelength and dilution level, high liposomes (tube 17-26) have an absorbance
around 0.37 to 0.46 and medium liposomes (tube 11-16 and 27-30) have an
absorbance of 0.18 to 0.36. The tubes after the 30th tube showed a significant increase
in the signal. These tubes were not analyzed because those contained high percentage
of free dye and very small percentage of liposomes.
0.5
Absorbance
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
10
20
30
40
Tube Number
Figure 3: Absorbance of liposomes at 532 nm after purification using Sephadex G50 column.
16
The liposomes were then purified from any remaining free dye using dialysis
membranes in a beaker containing 0.01M HEPES, 0.2 sucrose, pH 7.5. The dialysis
membranes containing the liposomes were allowed to stir in the beaker overnight.
Free dye not encapsulated by the liposomes filtered out of the dialysis membrane into
the buffer solution. To determine exact lipid concentration, Bartlett assay was
performed on the liposomes.
3.2.2
Bartlett Assay
Bartlett assay was performed to determine the amount of phosphorus in
liposomes where one mole of phosphorus is equal to one mole of phospholipids.
To make the phosphorus standard, 55.8mg of potassium phosphate dibasic was
dissolved in distilled deionized water in a volumetric flask. Water was added until the
total volume reached 100mL and the concentration of this solution came to be
3.2 mol of phosphorus per mL The standard was diluted to 16 nmol and these
solutions were treated in parallel with the samples. Fiske-SubbaRow Reagent was
prepared by adding 100mg of 1-amino-2-naphthol-4-sulfonic acid to 40mL of 15%
solution of sodium bisulfite while stirring for 30 minutes. The undissolved acid was
filtered through Whatman filter paper and stored in the dark.
20 uL of the liposome samples were pipetted into 30 mL test tubes in triplicate.
Phosphate standard were pipetted in triplicate into prelabeled test tubes: 0 nmoles
(0uL), 16nmoles (5uL), 32nmoles (10μL), 64 nmoles (20μL), 128 nmoles (40μL), 256
nmoles (80uL). These test tubes were put in the oven for 15 minutes to dry the liquid
17
and then were cooled to room temperature for 5 minutes. 1mL of distilled deionized
water was added to all tubes.
First, the phospholipids in the liposomes are acid hydrolyzed to inorganic
phosphate. This hydrolyzation was achieved by first adding 0.5mL of 10N sulfuric
acid (H2SO4) and vortexed for 10 seconds. Then these tubes were placed in the oven
for digestion to take place (175oC for 3 hours). And the tubes were cooled to room
temperature. 0.1mL of 30% H2O2 was added to the tubes and then tubes were
vortexed for 10 seconds. Test tubes were returned to the oven for 1.5 hours;
afterwards the test tubes had clear solution. 4.6mL of a 0.22% ammonium molybdate
was added to each tube to convert inorganic phosphate to phosphor-molybdic acid.
0.2mL of Fiske-StubbaRow reagent was added and tubes were vortexed for 10
seconds. The tubes were covered with glass marbles and placed in rack in boiling
water bath for 7 minutes.
The tubes were then quickly cooled in ice water bath to near room temperature.
This phosphor-molybdic acid had been quantitatively reduced to a blue colored
compound by amino-napththyl-sylfonic acid. The intensity of the blue color was
measured using a spectrophotometer at 824nm. The intensity was compared with the
calibration standards (Figure 4) to determine the phosphorus content and thus find the
phospholipids concentration.
18
Absorbance @ 825 nm
0.35
0.3
y = 0.0012x + 0.0072
0.25
R2 = 0.9975
0.2
0.15
0.1
0.05
0
0
100
200
300
nmol phosphorous
Figure 4: Calibration standards of the absorbance measured at 825nm for nmol phosphorous
content with triplicates of 3.2μmol/mL phosphate dibasic in volumes of 0, 5, 10, 20, 40, and
80μL.
The phosphorous content of the high liposomes were 103.2 nmol per 20μL of sample
and of the medium liposomes were 64nmol per 20μL of sample.
3.2.3
Streptavidin Coupling
DPPE-ATA, an activated lipid, was incorporated into the initial stages of
liposome preparation to allow coupling of streptavidin to the liposomes. For a final
mol% tag of 0.2 for streptavidin, 17.86nmol of streptavidin was allowed to react with
8.93μmol total lipids on the liposomes. Initially, streptavidin was derivatized with
sulfo-SMCC. To obtain 15 equivalents of sulfo-SMCC per streptavidin molecule
(268nmol sulfo-SMCC/mg streptavidin), 45.8 nmoles/μL solution of sulfo-SMCC in
DMSO (5.86 μL sulfo-SMCC in DMSO/mg streptavidin) was used. This mixture was
19
incubated for 2–3 h to derivatize the amino-modified nucleotide probes with
maleimide groups.
The ATA groups on the liposomes were deprotected by deacetylating the
acetylthioacetate groups on the surface of the liposomes, generating sulfhydryl groups.
100 equivalent of a 0.5 M hydroxylamine solution (pH 7.0 in 25mM EDTA, 0.1M
HEPES: 348mg hydroxylamine HCL, 104 mg EDTA, 238mg HEPES in 10 mL
deionized water) was added to the volume of liposomes. Then, for actual conjugation
of the streptavidin to the liposomes, the SH-tagged liposomes and the maleimidederivatized streptavidin were mixed and allowed to react on rotating mixer for 1 hour
at room temperature, then overnight at 4°C.
To quench the excess sulfhydryl groups on the liposomes and the unreacted
sulfo-succinimidyl groups on the sulfo- SMCC, 50 equivalents of 0.1M Nethylmaleimide (NEM) in PBS was added and mixed on rotating mixer for 4 hours at
room temperature. The tagged liposomes were purified from free reporter probe by
gel filtration using a Sephadex G50 column and subsequently by dialysis for overnight
using a 0.01 HEPES buffer, 0.2M sucrose, pH 7.5. Liposomes were stored in the dark
at 4 °C.
3.2.4
Antibody Immobilization
The 10μL of streptavidin conjugated liposomes were combined with 10μL of
100μg/mL biotinylated anti CD3 antibody in 1 to 1 volume ratio. This mixture was
allowed to be incubated at room temperature for 1 hour and stored in the dark at 4 °C
for up to 5 days.
20
3.3
Antibody Immobilization onto Magnetic Beads
The Streptavidin coupled Dynabeads MyOne coated with a monolayer of
recombinant streptavidin. Each mg of Dynabeads MyOne Streptavidin binds
approximately 300 pmol free biotin or 20 – 25 μg biotinylated IgG. 10mg/mL of
beads are concentrated in the stock solution. The concentration of beads in the stock
solution is 10 mg/mL and approximately 7-12x10^9 beads are in 1mL.
Conjugation of biotinylated antibodies to streptavidin coated
superparamagnetic beads was carried out following the manufacturer protocol. First,
2μL bead stock (10mg/mL) were washed two times in 1M PBS, 0.2 sucrose, pH 7
solution and resuspended in 10μL of the same buffer. 5μL anti-CD45-antibodies or
1μL anti-mouse-antibodies were added to the beads and the resultant mixture was
placed on a rotator for 30 min at room temperature to allow binding. Subsequently, the
beads were washed 3 times with 20μL 1M PBS, 0.2M sucrose, pH 7 solution and
resuspended in the same buffer to a final volume of 20μL with bead concentration of
1μg/μL. The immunobeads were stored at 40C for up to 5 days.
3.4
Red Blood Cell Lysis
The RBC lysis solution (ammonium chloride, ethylenediaminetetraacetic acid,
sodium bicarbonate) was combined with RBC sample in 3:1 buffer to blood volume
(30μL of buffer and 10μL of blood) and was mixed by inversion of the Eppendorf
tubes. The mixture was allowed to stand for 5 minutes in room temperature to allow
RBC lysis to take place. Then the Eppendorf tube was centrifuged for 20 sec at
21
14,000 rpm. The lysed supernatant was removed and the white blood cells were
resuspended in 10μL of 1M PBS, 0.2M sucrose, pH 7.
3.5
Microfluidic Device Components
Channels were fabricated on PDMS using a 4 inch master patterned silicone
wafer with a positive surface relief. The master pattern on the silicon wafer was made
using standard photolithography and dry etching technique and was provided by Dr.
Natalya Zaytseva. First, a mixture of PDMS prepolymer and curing agent in a volume
ratio of 7:1 was prepared and degassed under vacuum. One milliliter of this
prepolymer-curing agent mixture was then poured onto the silicone template. A level
surface silicone wafer was placed on top of the poured mixture. The obtained
sandwich structure (silicone wafer – prepolymer curing agent – silicone wafer) was
cured in an oven for two hours at 65°C. Afterwards, the assembled sandwich structure
was cooled and the PDMS film with the channel grooves (a replica of the design on
the silicone wafer) as shown in Figure 5 was peeled off the master.
500μm
100μm
Detection zone with magnet
34,000μm
Figure 5: Design and dimensions of the channel patterned onto the PDMS film. The channels
are 50μL in depth with the width of the detection zone 500μm and of the winding channels
100μm. The total length of the channel network was approximately 14 cm long with from
inlet to outlet length 34,000μm. The number of the straight channels in the serpentine is 40
(38 of the full length and 2 halves)
22
Final thickness of the PDMS film was ~170 m with the channel network
dimensions of ~50 m in depth, ~100 and ~500 m in width of the serpentine
channels and the detection zone, respectively. The total length of the channel network
was 14 cm and the length from inlet to the outlet 3.4 cm. There were 40 straight
channels in the serpentine with 38 full length channels and 2 half channels. The inlet
(~250μm) and outlet holes were drilled into the PDMS film using a high gauge needle
with a blunt end or a borer. Cross sectional and top view of the assembled
microfluidic device is shown in Figure 6.
Figure 6: Cross sectional view of the assembled microfluidic device. Two Plexiglas plates
house the microchannels created by placing the PDMS with the glass slide.
A glass cover slip was used to seal the microfluidic channels reversibly. The
slip was cut out from a microscope slide and cleansed with chromic acid cleaning
solution before being used in the device. A stainless steel tubing (with outer diameter
of 0.51 mm and inner diameter of 0.25 mm) was glued into one of the Plexiglas plates
at the locations lined up with inlet and outlet holes of the PDMS film. The Plexiglas
plates had a well in the area lined up with the detection zone in the PDMS film for
placement of magnet required for capturing magnetic beads. The rare-earth
neodymium-iron-boron magnet obtained from Grade N40, National Imports, Inc. was
23
used. A leak proof sealing between PDMS and glass substrates was achieved by
applying slight pressure by screwing the two Plexiglas plates with 6 to 8 screws.
3.6
Sample Introduction and Analysis
3.6.1
Droplet Assay
In an Eppendorf tube, supermagnetic beads, blood sample, and liposomes were
incubated for 30 minutes at room temperature in a shaker. After incubation, the tube
was placed on the magnet for 2 minutes. The supernatant was removed by aspiration
with a pipette while the tube remained on the magnet. Then the sandwich complex of
liposome-T lymphocyte-bead in the tube was washed with 1M PBS, 0.2M sucrose,
and pH 7 solution. Finally the sandwich complex was resuspended in 1μL buffer and
placed on a microscope slide for analysis and quantification.
3.6.2
Microfluidic Assay
First, continuous fluid flow through the channel network was established by
applying a positive pressure at the inlet using a syringe pump obtained from KD
Scientific Inc., Holliston, MA. The top of the steel tubing in the inlet hole was
connected to the syringe on the pump by Tygon tubing with an inner diameter of
0.5mm. The channels were prefilled with running buffer (1M PBS, 0.2M sucrose,
0.01% Triton X) at a slow flow rate of 2μL/min to prevent bubble formation. In an
Eppendorf tube, supermagnetic beads, blood sample, and liposomes were incubated
for 30 minute at room temperature in a shaker. Following the incubation, the mixture
was withdrawn into the tube that was connected to the syringe filled with the running
24
buffer. The end of the tube was placed back onto the inlet hole and the sample was
loaded into the microfluidic channel through at a flow rate of 5μL/min. The liposomeT lymphocyte-bead complex was captured by the magnet in the detection zone.
3.6.3
Fluorescence Measurement
Fluorescence of liposomes was visualized using a Leica DMLB microscope
(Leica Microsystems, Wetzlar, Germany). The microscope was set up with a 10/0.25
NA long working distance objective, the appropriate filter set of 540/25 nm band pass
exciter and 620/60 nm band pass emitter, and 100 W mercury illumination source.
The images of beads in the detection zone were obtained with a digital CoolSnap CCD
camera purchased from Photometrics, Tucson, AZ coupled to image acquisition
software (Roper Scientific Inc., Tuscon, AZ). The fluorescence was quantified using
Image ProExpress software (Media Cybernetics, Silver Spring, MD).
25
4.
RESULTS AND DISCUSSION
4.1
Determination of Background Binding
Initially, droplet testing was proposed as a way to measure the isolated
sandwich complex of liposome- T lymphocyte – beads. Before the actual cell
measurement, the liposomes and beads were tested for non-specific binding. Figure 7
below shows the pictures of the mixture of 1μL beads and 1μL buffer solution, 1μL
liposome and 1μL buffer, 1μL liposome and 1μL streptavidin conjugated beads, and
1μL streptavidin conjugated liposome and 1μL streptavidin conjugated beads.
Figure 78: CCD Image of negative control (1μL beads), positive control (1μL liposome &
1μL beads), liposome unspecifically bonded to beads, and streptavidinylated liposomes
unspecifically bonded to beads.
The mixture of 1μL beads and 1μL buffer was expected to have no signal and
was used as negative control. The mixture of unwashed liposomes and beads (the
positive signal) were expected to have the highest signal. Comparison of the liposome
and streptavidin conjugated beads versus the streptavidin conjugated liposome and
streptavidin conjugated beads showed that having streptavidin on both liposome and
beads liposome reduced the nonspecific binding of the two. Problem with reliability
came up with the droplet testing. Droplet on a microscope slide formed a meniscus
that produced a thick layer of liquid with freely floating magnetic beads. Because the
microscope cannot take 3 dimensional images, the thickness of the liquid created
26
layers of beads that could not be properly measured. Therefore, quantification was not
possible. Thus instead of using a droplet testing, decision was made to use a
microfluidic device was used to detect and isolate the liposome-sample-bead complex.
4.2
Optimization of the Microfluidic Device Operations
4.2.1
Optimization of Running Buffer
Initially, 1μL of streptavidin conjugated beads were tested in the microfluidic
device. Phosphate buffered saline + 0.2M sucrose solution was used as the running
buffer. When the microchannel was viewed through the X10 magnification of the
microscope, the beads were spread out at the neck of the channel where the 100μL
width channel opened up to the 500μL width capture zone. To solve this problem,
0.01% Triton X was added to the running buffer. When tested with the new buffer,
the magnetic beads all passed through the widening neck and congregate at the
detection zone.
4.2.2
Optimization of Buffer Flow Rate
The magnetic beads were captured on the detection zone within the
microchannel by an externally positioned magnet. The distance between the magnet
and the capture zone of the microfluidic channel determined the strength of the
magnetic field created by the magnet. This distance was determined by the depth of
the groove drilled in the Plexiglas housing and the thickness of the PDMS film. The
strength of the magnetic field decreased exponentially over distance. Hence, the
distance had a big influence on the flow rate; stronger magnetic field (obtained by
27
closer distance) allowed capturing of the beads at a higher flow rate. The thickness of
the Plexiglas plate between the magnet and the capture zone was approximately
400um.
The beads were introduced into the microchannels at a flow rate of 1μL/min.
The capture zone was view through the microscope as the buffer flow rate was
increased. When the flow rate reached 4μL/min, beads were pulled off from the
capture zone and washed away. To increase the maximum flow rate, the Plexiglas
housing was sanded to decrease the thickness. The final thickness was approximately
300um. The same test was repeated; the beads were introduced into the
microchannels and the buffer flow rate was slowly increased. The beads started
washing off the detection zone only when the flow rate reached 8μL/min. Because the
bulky sandwich complex would not withstand the higher flow rate than just the beads
could, all the tests from this point on used the flow rate of 5μL/min.
4.2.3
Optimization of Flow Time
The minimum flow time that was necessary for consistent signal generation
was determined. When the sample that contained the sandwich complex of liposomes
– T lymphocytes – beads and residual substances of unbound liposomes, beads, and
cells were introduced into the microfluidic device, a range of time was required for the
beads to be immobilized on the detection zone and then the residues to be washed
away from the detection zone. With the flow rate at 5μL/min and the volume of 10μL
(5μL of the sample and 5μL of the buffer) introduced to the device, approximately 2
minutes would be required for all the liquid volume had passed the detection zone and
28
maximum signal would be apparent. From that point, the signal would decrease and
the running buffer passes the detection zone and flushes away any unbound substance.
The signal would reach a steady state value when all the possible substances are
washed. The trend in the signal was observed over time to see the minimum time
required for the signal to reach a constant value.
Two different samples were introduced for determination of the flow time.
First a sample with streptavidin immobilized beads, blood, and streptavidin
immobilized liposomes were introduced to the microfluidic device. In this case, no
binding should occur and the signal obtained should be considered the inherent
background signal of the current device. Then a sample with 1μL immunobeads,
0.1μL blood, and 2μL immunoliposomes were used to determine the necessary flow
time. In this case, a sandwich complex should be captured at the detection zone and
produce a much higher signal than the first test.
In both cases, the time was measured from the point after the sample was
injected into the device at a flow rate of 5 L/min and washed with the running buffer
at a flow rate of 5 L/min. The results were as expected, i.e. a steady state signal was
obtained after about 10 minutes (which correlates to approximately 40 L of washing
buffer used in order to clean the capture zone from non-bound liposomes) and the
specific signal was about 3x higher than the background signal (i.e. 28.5 and 9.4,
respectively) (Figure 8). From this point, all tests were run for 10 minutes before the
signal was measured.
29
45
40
35
Signal
30
25
20
15
10
5
0
0
5
10
15
20
25
Time (min)
Figure 8: Determination of the minimum flow time needed to wash unbound substances.
Signal from sandwich complex (in pink) and 1μL liposome-1μL blood-1μL beads mixtures (in
blue) were measure over time at a flow rate of 5 L/min.
4.3
Testing of the Biosensor Components
4.3.1
Negative/Positive Control
To determine that the streptavidin immobilized liposomes were correctly
prepared and that antibodies were properly conjugated to immunoliposomes and
immunobeads, anti-mouse-antibodies were used. Anti-mouse-antibodies bind to any
anti-antibodies prepared from a mouse. Anti-CD3 antibody conjugated liposomes and
anti-mouse-antibody conjugated beads were prepared. The signal produced by the
immunoliposome and immunobead complex should be the maximum possible
intensity that would be detected and can be used as a positive control. Anti-mouseantibody conjugated beads were run without liposomes for negative control. The
positive control gave the intensity of 85.0 and the negative control showed no
fluorescence.
30
4.3.2
Non Specific Binding Test
To understand the origin of the non-specific binding observed in the previous
Section 4.1, different combinations of streptavidin immobilized beads, streptavidin
immobilized liposomes, blood, lysed blood, immunobeads, and immunoliposomes
were mixed and ran (Figure 9).
Streptavidinylated beads and streptavidinylated liposomes produced a signal of
24.4. However, when streptavidinylated beads were mixed with immunoliposomes,
the signal nearly quadrupled to 88.2. The freely floating biotinylated anti CD3
antibodies from the immunoliposome sample interacted with the streptavidin on the
magnetic beads and caused a high signal that matched the positive control value from
Section 4.3.1.
When the beads were coupled with anti-CD45-antibodies and then mixed with
the immunoliposomes, the signal decreased by 50% to 46.0. The signal could be due
to the uncoupled streptavidin on the beads or the interaction between the antiantibodies CD3 and CD45. However, the beads were coupled with the antibody
concentration recommended by the company for saturation and should not have any
free streptavidin sites.
To see if the non-specific binding was owed to unsaturated binding sites, and if
the signal could be reduced, a blocking reagent (0.015% casein, 0.5% PVP, 0.25%
gelatin, 1X TBS, 0.002% Tween-20) was used to treat the immunobeads prior to
testing. When the treated immunobeads and immunoliposomes were captured, the
signal came to be 33.0. The signal decreased by 33% from the untreated
immunobeads/immunoliposome sample. Some of the background signals were from
31
the biotin on the mobile antibodies nonspecifically binding to free streptavidin on the
beads, but some signal must be due to the interaction of the antibodies. From this
point, all tests were run with immunobeads treated with blocking solution.
100
90
80
88.2
60
50
40
45.9
30
33.0
20
immunobeads +
immunoliposomes
beads +
immunoliposomes
0
24.4
beads +
liposomes
10
blocked
immunobeads +
immunoliposomes
intensity
70
Figure 9: Comparison of the signal produced by beads/liposomes, beads/immunoliposomes,
immunobeads/immunoliposomes, and treated immunobeads/immunoliposomes.
32
4.4
Standard Dose Response Curve
For this project, exact count of the blood cell was not obtained. Instead
fractions of the blood sample (1μL) were captured (Figure 10) to create the dose
response curve using whole blood and lysed blood samples to understand the possible
detection limit as seen in Figure 11.
(a)
(b)
(c)
(d)
Figure 10: Image of the capture zone for whole blood sample. From left: (a) Positive control
(antimouse antibody beads-immunoliposome), (b) assay with 100% 1μL blood, (c) assay with
10% 1μL blood, (d) assay with 1% 1μL blood
Blood was diluted and used in the assays, as 100% blood sample
(concentration fraction 1), 10% blood sample (concentration fraction 0.1) and 1%
blood sample (concentration fraction 0.01) diluted in the same solution as the running
buffer (PBS, 0.2 sucrose, 0.01% Triton X) right before testing. For a concentration
fraction of 2, 2 L of 100% blood were analyzed. For both sets of analysis, a clear
dose response could be observed, i.e. the signal increased with increasing blood
fraction. The maximum signal obtained for whole blood with 2 L of analyte, the
signal was 75.7, approximately 10% below the positive signal. This difference in
signal showed that the liposome concentration was not saturated by the CD3 cells and
that enough liposome concentrations were used for testing.
33
blood
lysed blood
positive control
blocked immunobeads/immunoliposomes
90
80
70
intensity
60
50
40
30
20
10
0
0
0.5
1
1.5
2
concentration fraction
Figure 11: Standard dose response curve of the lysed blood sample and whole blood sample.
A positive (red) obtained from testing of anti-mouse antibody conjugated beads with
immunoliposomes were drawn to show the maximum signal that can be produced by 1μL of
liposomes. Non specific signal produced by blocked immunobeads/immunoliposomes (green)
was graphed for comparison.
However, for the lowest blood concentration fraction (0.01), the signals
increased, which was assumed to be due to non-specific binding of immunoliposomes
and immunobeads. Without enough cells bound to the beads, immunoliposomes had
more available beads to interact with. Furthermore, in the case of lysed blood (1μL),
the signal remained at all times below, whereas the signal from whole blood (1μL)
remains above, the background signal discussed in Section 4.3.2. The differences in
the signal were closely observed in Figure 12.
34
70
60.1
intensity
60
50
33.0
40
25.7
30
20
10
blocked
immunobeads +
immunoliposomes
lysed blood
whole blood
0
Figure 12: Comparison of the signals of whole blood sample (blue) and lysed blood sample
(purple) and blocked immunobeads and immunoliposomes (beige).
The signal from the test of 1μL immunobeads + 1μL whole blood + 1μL
immunoliposomes was determined to be 60.1. The signal from the test of 1μL
immunobeads (anti-CD45-antibody) + 1μL lysed blood + 1μL immunoliposomes was
25.71, less than half the value of the whole blood sample. The large difference in the
signal strength at same concentration of T-lymphocytes in the sample suggests the
possibility of background signals from the unwashed red blood cells. Because red
blood cells emitted red fluorescence as SRB, the microscope could not distinguish the
difference and would detect signal from both the cell and the liposomes.
If the non-specific interaction was consistent with or without the presence of
the blood sample, the signals obtained from testing blood samples would be higher
than the signal obtained for the non specific binding. However, this was not the case;
the signal from the lysed blood sample was lower than the non-specific signal
35
produced by the interaction of the blocked immunobeads and immunoliposomes
without the presence of blood sample. This result suggested that the specific binding
of the T-lymphocytes onto the magnetic beads reduces the nonspecific binding of the
free immunoliposomes. Prior to incubation and injection into the microfluidic device,
the immunoliposomes (anti-CD3-antibody labeled) were added after the immunobeads
(anti-CD45-antibody labeled) and blood samples were pipette mixed. Binding of the
white blood cells could be decreasing the free beads available for non-specific binding.
In healthy humans there are approximately 4,500 to 10,000 white blood cells
(CD45 labeled cells) per 1μL of blood. Of those white blood cells, 20-50% are
lymphocytes with 800 to 2,200 T-lymphocytes per 1μL blood. According to the dose
response curve, the 0.01 fraction would have T-lymphocyte count between 8-22 and
0.1 fraction with 80-220 cells. Hence, according to the dose response curve, the
detection limit would be at or below the 0.1 concentration, thus 80 – 220 cells could
be detected.
36
5.
CONCLUSION
Microfluidic biosensor using immunobeads as capture probes and
immunoliposomes and reporter probes for detection and quantification of Tlymphocytes have great potential for application in the health care system. The tests
performed in this study will give better direction to future testing of this biosensor for
immunophenotyping.
Numerous tests were accomplished to understand the efficiency and reliability
of the immunoliposomes as signal probe and immunobeads as capture probe.
Preliminary studies of the non-specific bindings of liposomes and magnetic beads
were observed using a droplet test. However, varying thickness of the drops produced
inaccurate and unreliable signals and instead a microfluidic device was used. Initially,
the device was optimized for operation. Running buffer was modified to prevent
sticking of the beads to the walls of the microfluidic channels. Then the optimum
buffer flow time was determined to be 10 minutes to allow minimum but sufficient
time for washing of unbound cells and liposomes.
Following the operation modification, the biosensor components were tested.
Coupling of anti-antibodies almost doubled the nonspecific binding signal of
liposomes and magnetic beads from 24.4 to 45.9. Using blocking reagents to treat the
immunobeads prior to combining with immunoliposomes reduced the signal by 33%
to 33.02. The speculation was made that some non-specific (background) signals were
due to the interaction of the anti-antibodies and not just biotinylated or
streptavidinylated ends.
37
The background signal from the non-specific binding of red blood cells was
decreased by lysing the whole blood sample before testing. The limit of detection of
the microfluidic biosensor was determined to be at or below a whole blood fraction of
0.1, which can be correlated to 80 – 220 T-lymphocytes per L of blood sample.
Variability of the results could have resulted from the instability of the blood
sample. Blood samples are normally stable up to 48 hours after collected. For
preliminary experimental purposes, the blood samples could not be obtained as
frequently and the experiments were run on blood older than 48 hours. Further
optimization of the proposed design is required including modification of sample
injection method to decrease the amount of sample lost to the surface where the
sample mixture is placed before withdrawn into the tubing and injected into the device,
of streptavidin concentration on liposomes to decrease non-specific binding, and of
working buffer used in blood and bead dilution. Fresh blood sample should be used to
check the accuracy of the values and exact count of T-lymphocytes should be obtained
to determine the exact limit of detection.
However, the data shown here prove the principles of the micro-biosensor for
T-lymphocyte detection. Success of this immunophenotyping biosensor will provide
portable and cheap device with minimum human handling that can be used for Tlymphocyte enumeration. Microfluidic biosensor has great potential for testing in
resource-limited settings.
38
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