How to Poke a Cell
Vo-Dinh, T. “Nanobiosensors: Probing the Sanctuary of Individual Living Cells.” Journal of Cellular Biochemistry Supplement, 39:154–161 (2002).
Summary:
A biosensor is a measuring device, consisting of a transducer and a probe with a bioreceptor (an antibody or a protein). When a given chemical species reacts with the bioreceptor, the transducer converts the resulting change into a quantifiable signal. Previous research has shown that biosensors are useful in fiberoptics. A nanobiosensor (nanosensor) has all the properties of a biosensor with the added benefit of nanoscale physics. When light is sent through the nanosensor, it proceeds down the length of the sensor until it cannot proceed any further. In large-scale optical fibers, photons are able to exit from the sensor tip. But in nanofibers, the diameter of the tip (about 50 nm) is much less than the wavelength of light used. Photons cannot escape. They simply travel as far as possible and then emit evanescent fields which continue through the remainder of tip. These fields interact with any species of interest that lie extremely close to the sensor tip. 
The nanofiber can be fabricated in two ways: the “heat and pull method” and chemical etching of glass fibers. In the first method, a larger silica optical fiber is clamped in a specialized device as a laser beam is fired at it. The device starts pulling the fiber apart. This tension combined with laser heating results into two nanofibers with a very fine tip. One side of the tip is coated with a layer of silver, aluminum or gold to prevent light leakage during excitation. Then, antibodies are immobilized onto the other side of the tip, constituting the bioreceptor.
In this paper, anti-benzopyrene tetrol (BPT) was used. BPT is a chemical marker for exposure to benzo(a)pyrene (BaP), a chemical carcinogen that is widely present in the environment. Clone 9 cells, from a rat liver epithelial cell line, were grown in bovine serum and glutamine media. After passaging cells once, they were allowed to grow to 60% confluence before BPT was added to the culture. Using bright field microscopy, a nanofiber tip was positioned directly outside a chosen cell in the culture dish. Laser light was sent through the nanofiber and a control reading was taken. Then the tip was moved into the cell, piercing the cell membrane but not the nuclear envelope. Obtained readings were passed into a photomultiplier tube for detection. Using cultures with different concentrations of BPT to obtain an exponential regression, the concentration of BPT in this particular section of the cytoplasm of a particular cell was measured.
Problems:
A quickly identifiable problem might be cell viability after this procedure. This paper outlines a subsequent experiment done where probes were left inside cells for 5 mins and then removed. The cells were allowed to rest and actually underwent mitosis after probing event. Routinely, in gene transfer experiments, cells are probed by micropipets and retain function. Further research should be done to determine any changes in extracellular communication, signaling and antigen-antibody binding (due to disruption of cell membrane).
Relevance:
Traditional dye-based microscopy techniques are dependent on dye concentration and the ability of the cell to transport the dye to the region of interest. Often cells are also fixed before injection with dye, which might kill the cells or change their internal connections. With optical nanosensors, cells are alive and detection is only dependent on analyte concentration. The excitation light leaving the nanosensor tip is localized so no extraneous signals interfere with readings. Once the probe is inserted and measurements taken, the probe can be removed, unlike fluorescent dyes. The minimal invasiveness of the technique is of key significance. Measurements of biochemical pathways taken from a functioning cell might prove more useful than Western blot and SDS-PAGE measurements taken from cell lysates. The lysis process might destroy key intermediates in the investigation of a new chemical pathway. Also, cell lysis cannot tell us a molecular concentration in the nucleus vs. in the cytoplasm vs. in the ER. But nanobiosensors can.
Further work:
Nanobiosensors have been used in MCF-7 breast cancer cells to detect the caspase-9 activity, which leads to the release of cytochrome c and apoptosis-inducing factor (AIF). Nanosensors were affixed with LEHD-AMC, the substrate of caspase-9. By measuring capsase-9 levels, researchers were able to distinguish between cancer cells that were “normally” dividing and cancer cells that were beginning apoptosis, long before apoptosis markers appeared. Because of this nanobiosensor technology, apoptosis can be monitored in vivo at the single cell level (Vo-Dinh 2008).
10 comments:
I found this to be a fascinating use of biosensors and a great alternative to many of the cell biology techniques currently used. It is true that immunofluorescence and other methods utilized often damage the cells in the process of examining them and may impede our abilities to understand the greater picture of cell pathways and activities. It seems to be a good method for laboratory work and probing on a very short time scale. I wonder however, if it can be useful in long-term use, for example, as an in vivo blood glucose monitoring system. Was this at all mentioned as a possible avenue of research in the paper? One of the biggest problems with biosensor is biofouling from the host response to the foreign material in the body. As proteins begin to adsorb to the material surface, cells adsorb and can ultimately interfere with the sensors ability to read information from its microenvironment. I see great potential in these nanobiosensors for long-term clinical use in diabetes and cancer patients if the issues of biocompatibility can be addressed.
This idea of bionanosensors could also aid in research on an individual cell-basis to determine differences in environments within a cell culture. Chemotaxis could be better identified by testing individual cells as they move to see if they are responding to a chemoattractant or moving randomly. My group did a chemotaxis assay and it was difficult to determine if cells were moving due to the chemoattractant or if the movement was a random expansion of the colony. Further research with this bionanosensor could show the difference in each environment based on a cell's response. It is interesting that in this paper, readings were taken both outside and inside the cell, which could further help the understanding of signals sent from cell to cell.
Joy: You are absolutely right. Protein fouling is the key problem with biosensors. Here, they are immobilizing antibodies on the probe, which means any protein in the vicinity can bind to the probe as well. The researchers in this paper did multiple repeats of their experiments and disposed of probes in between experiments. They even stated that this idea is a one-use-only sensor, like glucose strips in the pin-prick blood glucose sensors. Because of this limitation, I don't think that this probe has a future in long-term in vivo sensing. But I can easily see how changing the material (using nanophase materials that resist protein adsorption) can open up possibilities in long-term clinical use.
Sydney: Definitely. The biggest contribution this nanoprobe can make is at the individual cell and cellular environment level. As for the chemotaxis experiments, this probe would help. But cellular signaling in response to a chemoattractant is so complex. Functionalized antibodies on this probe tip would have to be specific to a compound only made in response to a certain attractant. Signaling paths are usually interconnected, so that can prove difficult. If the molecular biology complications are resolved, then absolutely, the nanobiosensor is perfect in cell environment testing applications.
Hey Lavanya,
The use of biosensors to "poke" the cell is such an interesting idea and I did immediately wonder if the cells would be viable after getting "poked," but you addressed that concern under the problems section of your report. What I am now pondering kind of ties in with Joy's comment. Since proteins can bind to the sensor, do you think it reasonable to resurface the probe to prevent unwanted protein adhesion, especially since the device is so small?
Thanks for sharing such an interesting article.
-Robert
Hi Robert,
You're right. It is possible to adsorb compounds to the surface to prevent protein adsorption. Or they could even change the probe material properties. But this would increase the cost of the system. The paper touted this idea as a cost-effective, relatively non-messy, accurate way to measure signaling or presence of certain proteins in and outside a cell. Attaching a compound (like a polymer that prevents fouling) would have to be done carefully to not cover up the area where the antibody must bind. And there is a region where light must hit; they cannot functionalize anything there.
And we haven't even considered the size scale. How large is an attached polymer or compound compared to the literally ten's of nanometers or so length scale of the probe tip? Assuming the cell "poking" doesn't impede viability, is it safe to insert a probe with other polymer compounds that usually wouldn't be found inside a cell? I don't have the answers to these questions. But definitely something to look into. Great point.
This tool of biosensors is quite fascinating for studying cellular level activity both inside and outside the cell. However, I am also highly concerned about the cell viability after poking the cell with a nanofiber tip because we are dealing the nanoscale activity, and the ability to really confirm that cell viability is not decreased or that the poking does not disturb original cell activity is difficult. For instance, if we want to measure intracellular activity using this poking method, how do we know if extracellular fluid would enter the cell after poking, which will disturb the signal readout? There are less invasive methods like the use of microfluidic devices for specific studies on ion channels of cell to cell intactions through patch clamp, gap junction, neurons on transistors, and finally neural probes. You mentioned the advantages of cell poking to detection through immunofluorescence and other methods, yet would you believe that they outweigh the potential damage and disturbed signal of nanofiber probes?
I found this paper idea very interesting! I would like to learn more about the viability of the cells that undergo this method. You state that this poking technique is not invasive, and that the cells could undergo mitosis after probing. I think further tests would need to be run to show that the cells could undergo all activity it normally does. Is mitosis enough of a confirmation that cell activity isn't interfered with by this probing action? I think the point of traditional dye based methods is that they do not touch the individual cells and would be less invasive because it is through dye.
Hi Lavanya, this research is pretty incredible. Was there any information given on how the researchers exercised such great control with the tip such that it pierced the cell membrane but nothing further or is this not as complicated as it seems to me? Also, in the test for cell viability after the probes are injected, did the paper mention the material of the probe having any effect on this at all?
Suruchi:
The researchers did not vary the probe material at all. That definitely should be in the works for future studies. As for the mechanics of the "poking", they didn't really discuss it. But I assume that the researchers are well-trained in this field and they observed their own "poking" actions with a microscope as the probe entered the cell. This type of work is related to how researchers remove a cell's nucleus and insert a new nucleus (ex. cloning).
Audrey and Cindy:
You both address the same issue of cell viability after poking. It is the issue of most concern in this experiment. I do think more study is needed on this topic. That being said, just because the cells undergo mitosis after "poking" does not mean that processes (like signaling which depends on ECM and membrane components) were not disturbed.
Audrey, you were talking about intracellular fluid leaking in after the "poking". Remember that this is a nanoprobe so the hole it creates is so small that the amount of fluid entering the cell would be very small. Also, the probe can be positioned at quite a distance away (in cellular terms) from the point of entry to avoid signal contamination. As it stands now, without further research into this probe and its effects, the risks to a cell outweigh the benefits. But that's the beauty of further research - one paper doesn't solve a entire field's worth of problems. But it's a start.
Also, Cindy, you are right that proof of mitosis doesn't mean cell function is normal after probing. I completely agree with you. But fluorescent labeling and further microscopy detection is very damaging to cells. If you want proof, check out this paper:
Dixit R and Cyr R. "Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy." The Plant Journal v 36, issue 2, pp.280-290.
It talks about how light intensity causes irreversible damage to the cell's signaling paths. Also, oxygen radicals are formed which can attack cell membranes. When we use fluorescent molecules with cells, we can't use the cells for anything else. They are essentially killed by the process. That's why an alternative is needed. This probe is an alternative, albeit one in its infant stages.
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