Biomod/2011/HKBU/NBgamers:Project

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NBgamers (Team of NanoBiotechnology)
 

Project

Contents

  1. Project Title
  2. Abstract
  3. Design
  4. Results and Discussion
  5. 3D Animation
  6. Material and Method
  7. Potential Diversion

Project Title

High Throughput and Ultrasensitive Beta-Amyloid based Nanosensor for the Detection of Biomolecules


Abstract

We develop a simple and efficient technique using self-assembling beta-amyloid (Aβ) nanofibrillar sensor for ultrasensitive and pretreatment-free detection of biomolecules. As a proof-of-concept, small DNA (ca. 20 nt) is used as target analyte model for demonstration. Herein, Aβ fibrils conjugated with complementary DNA probes are applied as the detection template to capture (by hybridization) and preconcentrate the target DNA in solution. With the aid of total internal reflection fluorescence microscopy (TIRFM), two types of fluorescence signals would be collected. YOYO-1 iodide, which is a DNA bis-intercalating dye, is used to label the DNA hybrids for quantification; while quantum dots of different emission wavelengths are tagged and utilized to differentiate various probes conjugated on the fibrillar sensors, and hence achieving simultaneous multiplexed detection of biomolecules. In our assay, each well-defined fibril serves as an individual sensor, and thus one may perform numerous detection assays in parallel. In summary, this design offers fast yet accurate detection of small DNA in high-throughput manner without the need of sample enrichment. It also brings insight in the development of novel biomaterial-based sensors in nanoscale.


Design

Structure

1. Preparation of biotinylated beta-amyloid fibril as biosensing template

Beta-amyloid (Aβ) peptides self assemble into Aβ fibrils once they misfold. Synthetic Aβ are commercially available and the fibrillation conditions of Aβ is also well-established by many research groups. In order to adapt it as one building parts of a sensor, the native Aβ monomers are co-incubated with monomeric biotinylated Aβ to form biotin-functionalized fibrils. The morphology of the fibril formed by native and biotinylated monomers is found to be the same as the native fibrils (~10 nm in diameter and few microns in length). This confirms that the self-assembled fibril is now ready to be manipulated as a biosensor template.


2. Conjugation of streptavidin linker on beta-amyloid fibril

Quantum dot labeled streptavidin (commercially available) is attached onto the fibril by biotin-streptavidin conjugation. Quantum dot (QD) is a highly fluorescent nano-particle and different QDs have different emission colors of sharp peaks. Making use of this unique property of QDs, one can thus fluorescently label the fibrillar sensor with various colors respectively. [see multiplex detection]


3. Attachment of biotinylated DNA probes on amyloid biosensor

As a proof-of-concept for short nucleic acid detection, short sequence single-stranded DNA (15 nt) probe is particularly chosen in this project. The commercially available biotinylated DNA probe is then attached to the fibrillar sensor by binding to previous QD-labeled streptavidin as illustrated in the schematics. After that, the detection of target DNA molecule is achieved by specific and complementary hybridization between the probes and targets.


Project Scheme

Schematic of β-Amyloid based nanosensor (Click to enlarge.)


Highlights

1. High throughput

Under the ultrasensitive detection of total internal reflection fluorescence microscopy, single beta-amyloid fibrils can be resolved with high resolution. Herein, each fibril is independent from each other, and hence, each of them is served as an individual sensor in our experiment! Think of it, if you have 100 fibrils in a single fluorescence image, you have 100 sensors already!

2. Preconcentration

In typical single-molecule microscopy detection, molecules were freely diffused in the detection volume and therefore visualization and measurement of fluorescence intensity of those molecules is difficult.

Here in our novel assays, the biotinylated probe was immobilized on the Aβ (1-40) fibril, target will be entrapped and preconcentrated on a linear restricted region of fibrils instead of diffusion free in solution. You can now easily found your target analyte, preconcentrated online, and measure its intensity easily!

3. Pretreatment free

Traditional DNA quantification assay may either be semi-quantitaive or required sample enrichement in order to achieve a significant signals to be detected. Some assays may even need to modify the target analyte with fluorescence dyes or nanoparticles to generate signals. Now in our assay, all of them is not needed!

Our sample is pretreatment-free, we can detect our target analyte without the need of sample enrichment. Make it easy!

4. Ultra sensitivity

In our experiment, target analytes are being preconcentrated while our detection microscopy system, TIRFM, can bring as a very high signal-to-noise ratio. Our detection sensitivity is extremely high! Even without the need of sample enrichment, we can still able to quantify short DNA/RNA sequences with a picomolar concentration

5. Low cost and time saving

Time is money! Everybody wants to work fast with a low cost! Our experiences tell it takes only 2 hours to complete a whole trials of short DNA quantification and calibration experiments with high-throughput and good sensitivity which you have never experienced! Roughly estimating, it takes you less than 1 USD for each trial!


Results and Discussion

Highly Sensitive and High-throughput Detection of Target Molecules

Single-stranded nucleic acid targets are captured by complementary nucleic acid probe immobilized on the sensor fibrils as described in the project structure.

To visualize and quantify target sample, a highly selective nucleic acid fluorescence labeling dye YOYO-1 is utilized. YOYO-1 has high affinity to double-stranded DNS helix over single-stranded. Once it binds to double-stranded, it fluorescence intensity will be enhanced significantly (~1000-fold). In this case, any free YOYO-1 in the solution will be regarded as "silent".



Figure 1: Sensors alone without target in the presence of YOYO-1

Figure 2: Sensors and targets in the presence of YOYO-1

Quantification of Target Molecules

Quantification of target is achieved by measuring the resulted YOYO-1 intensity. The concentration of targets should be proportional to the fluorescence intensity emitted by YOYO-1.


Monoplex Detection

Conduct the detection using integrate sensor fibrils in a flow cell platform.


Figure 3: Flow cell


Perform the detection under TIRFM system.


Figure 4: Microscope

As demonstrated in our detection scheme, the target DNA molecules, instead of diffusing around, are online-preconcentrated into a well-defined nano-fibrillar structure. It implies that this detection assay is of high sensitivity and precision. Unlike traditional analytical methods, no sample enrichment and amplification is required.


Multiplex Detection

Sensors contain different probes are integrated into a single platform for multiplex detection. QDs of different colors are used to distinguish different probing sensors.

Choose QD-streptavidin conjugates of different emission spectrum to label each kind of sensor. Then, select the respectively fluorescence signal from each kind of QD by corresponding optical filter.

Obtain the location of one specific kind of sensor according to selected QD signal (different colors), and then YOYO-1 intensity of that sensor can be measured for quantification as described previously monoplex detection.

Demonstration of multiplex detection for 2 targets using QD 565 (red color) and QD 625 (green color):

Prepare two different QD labeled sensors with different specific probes:

The fluorescence signals from QD 565, QD 625 and YOYO-1 are collected respectively under fluorescence microscope by using appropriate optical band-pass filters.


Figure 5: Image of QD 565 labeled fibrils from filter 1 (with pseudocolor)

Figure 6: Image of QD 625 labeled fibrils from filter 2 (with pseudocolor)

Figure 7: Image of fibrils with YOYO-1 from filter 3

The fibril sensor capturing target 1 and target 2 are located respectively with our developed program. The fluorescence intensity of YOYO-1 is measured for quantification of each target.


Data Analysis

The raw image obtained from fluorescence microscope is converted into fluorescence intensity for quantification purpose. Several steps are involved and a program is developed for automatic processing.

Protocols for data analysis: (see: programming code)

Step 1. Locate the fibrils

The raw image is transferred into binarized form in order to locate the fibril, therefore the background noise could be eliminated. The "bright" fibrils are regarded as our detection region.


Step 2. Find the background intensity around each of the fibrils

With the locations got from step 1, back-trace the sensor fibrils in the raw image and find the surrounding background intensity of each individual fibril. The average fluorescence intensity of the background is determined.


Step 3. Measure the net fluorescence signal from the YOYO-1 labeled hybrid

Signal intensity in chosen area in which contains target sample is then subtracted by the background intensity to get the "net" fluorescence intensity of YOYO-1 per pixel in detection region.


Step 4. Quantification of target with calibration

Target DNA molecules of various concentrations ranging from 250 pM to 5 nM were determined with our developed assay. A linear relationship (R2 = 0.998) between target nucleic acid concentration and YOYO-1 fluorescence intensity (obtained from step 3) is established as shown in the following calibration plot. These results demonstrated that our detection assay is of high precision and high sensitivity (pM).


Relative intensity of YOYO-1 versus target (poly A, 15nt) concentration

Fit the YOYO-1 fluorescence intensity measured from sample target into calibration curve to obtain the target concentration.


3D Animation


Material and Method

Experimental Procedures

Preparation of cover glasses

All coverslips were pre-washed prior to experiments.

  1. No.1 22 × 22 square mm cover glasses (Corning, NY) were successively sonicated in absolute ethanol, sodium hydroxide (NaOH) solution, glacial acetic acid and distilled water successively.
  2. The cleaned coverslips were dried completely at 140 °C oven for approximately 15 minutes and stored for future usage.

Preparation of Aβ1–40 fibrils for seeding
  1. Monomeric Aβ1–40 (Invitrogen) Aβ1–40 was prepared by dissolving 1 mg of Aβ1–40 monomers in 400 μL ice-cold 0.02 % ammonia solution and stored at −80 °C until use.
  2. Stock solution of Aβ1–40 was diluted to 50 μM with phosphate buffer (50 mM sodium phosphate, 100 mM sodium chloride, pH 7.4). The mixture was incubated in water bath at 37 °C with gentle shaking for 20 hours.
  3. The resultant fibrils were sonicated for 5 seconds thrice and used as seedings in the later experiments for the seed-mediated fibrillation.

Preparation of biotinylated Aβ1–40 fibrils
  1. Stock biotin-Aβ1–40 monomers (Anaspec, CA) were diluted with 0.02 % ammonia solution to 100 μM.
  2. Biotinylated-Aβ1–40 fibrils were prepared by mixing monomeric biotin-Aβ1–40 and Aβ1–40 in a molar ratio 1:4, 1:9, 1:19, 1:49 respectively, with a total Aβ concentration of 50 μM.
  3. 0.87 μg/mL of prepared Aβ1–40 seeding was added to the solution. The peptide mixture was then incubated at 37 °C for 60 minutes.

Immobilization of amyloid fibrils on the surface of flow cell
  1. Sealed flow cell was prepared by combining two pre-cleaned cover glasses with double-sided adhesive tapes with a channel width of approximately 3 mm each.
  2. 10 μL of diluted amyloid fibrils and buffer solution was flowed into the flow cell.
  3. Solutions in excess were withdrawn at the outlet with Kimwipes based on capillary force such that fibrils were stretched under the capillary force.
  4. 4. The flow cell was placed under the home-built prism-type total internal reflection fluorescence (TIRFM) system for imaging.

Optimization of fibril density and fibril co mposition
  1. Fibrils were immobilied on the surface of flow cell as described in the previous section.
  2. 10 μL of Qdot 625 streptavidin conjugate (QD625-Stv, Invitrogen) solutions was flowed into the channel after the flow of phosphate buffer and PBS buffer with 1 % of bovine serum albumin (PBS-1% BSA).
  3. The sample was incubated at room temperature for 30 minutes before imaging.
  4. The flow cell was placed under the home-built TIRFM system for imaging.

Detection of DNA sequences with biotinylated Aβ1–40 fibrils
  1. Fibrils were immobilied on the surface of flow cell as described in the previous section.
  2. Excess QD625-Stv were then added into the channel, incubated for 15 minutes and successively washed with buffer.Herein QD625-Stv was added for localization of fibrils in the images.
  3. Excess biotinylated-poly(T)15 probe (5’-Biotin-TTT TTT TTT TTT TTT-3’, Invitrogen, HPLC-purified) were added into the channel and incubated for 15 minutes to saturate the available Stv sites.
  4. The channel was then washed with phosphate buffer. Target poly(A)15 (5’-AAA AAA AAA AAA AAA-3’) of different concentrations were added into the channel, hybridized for 30 minutes at r.t. for detection and calibration respectively.
  5. YOYO-1 iodide (YOYO, Invitrogen) was finally added to label and produce fluorescence signals from the hybridized DNA duplex.
  6. The flow cell was placed under the home-built TIRFM system for imaging with the excitation of 488 nm laser.

Multiplexed Detection of DNA sequences with biotinylated Aβ1–40 fibrils
  1. Fibrils were immobilied on the surface of flow cell as described in the previous section.
  2. Excess QD625-Stv were then added into the channel, incubated for 15 minutes and successively washed with buffer.Herein QD625-Stv was added for localization of fibrils in the images.
  3. Excess biotinylated-probe A were added into the channel and incubated for 15 minutes to saturate the available Stv sites.
  4. Additional fibrils were immobilized on the surface of flow cell.
  5. QD565-Stv and biotinylated–probe B were added successively to the channel with a similar procedure as described in (2) and (3).
  6. YOYO-1 iodide (YOYO, Invitrogen) was finally added to label and produce fluorescence signals from the hybridized DNA duplex.
  7. The flow cell was placed under the home-built TIRFM system for imaging with the excitation of 488 nm laser using 3 different filter cubes.
  8. Herein, fluorescence signals from QD625-Stv (by using HQ625/20 filter) and QD565-Stv (by using HQ565/18 filter) were monitored to distinguish the location of fibrils immobilized with probe A and probe B respectively; while fluorescence signals from YOYO (by using HQ535/50) were measured for quantification purpose.

Instrumental Configurations

Total Internal Reflection Fluorescence Microscopy (TIRFM) imaging system
  1. Microscope: An inverted Olympus IX-71 microscope (Olympus, Tokyo, Japan)
  2. Objective: 60× (1.45 NA) oil-immersion objective (PlanApo, Olympus)
  3. Laser source for fluorescence excitation:
    1. 445 nm diode laser (50 mW, LQC445-40E, Newport, USA): for the excitation of thioflavin T (ThT) labeled Aβ1–40
    2. 488 nm cyan laser (50 mW, CMA1-01983, Newport, NJ): for the excitation of QD625-Stv, QD565-Stv and YOYO
    3. Incident angle of the laser beam: 66°(to achieve total internal reflection and generates evanescent field)
  4. Optical filters:
    1. HQ 535/50 (Chroma Technology Crop., USA): for observing YOYO fluorescence
    2. HQ 625/20 (Chroma): for observing QD625-Stv fluorescence
    3. HQ 565/18 (Chroma): for observing QD565-Stv fluorescence
  5. Detector: Electron-multiplying charge coupled device (EMCCD) camera (PhotonMax 512, Princeton Instrument, Princeton NJ, USA)
    1. Exposure time: 100 ms
    2. Amplification gain: 4000
    3. Accumulation: 10
    4. Image capturing software: WinSpec/32 software (Version 2.5.22.0, Downingtown, PA, Princeton Instruments)

Potential Diversion

Other self assembly peptides

To form a sensor template, the material isnot restricted to beta amyloid only, all self assembly peptide with fibril-likemorphology can serve this purpose.


Biomolecular detection other than nucleicacids

Not only this detection method can be usedto detect nucleic acid, by changing the probes, detection of other biomoleculescan be achieved as well. For example, a biotinylated antibody probe can detecta specific antigen target.


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