Cadmium-Free Quantum Dot-Based Biosensing System for Matrix Metalloproteinases

REU Student: Lee AmayaGraduate Student Mentor: John PlumleyFaculty Mentor: Dr. Marek Osinski

Key to AbbreviationsCNS – central nervous system; ELISA – enzyme-linked immunosorbent assay; IHC – immunohisto-chemical; MMP – matrix metalloproteinase; MMPI – MMP inhibitor; MPTMS – 3-mercaptopropyltrimethoxysilane; NP – nanoparticle; ODE – octadecene; PL – photoluminescence; PLE – photoluminescence excitation; QD – quantum dot; QE – quantum efficiency; SA – stearic acid; TIMP – tissue inhibitor of metalloproteinase; DLS - dynamic light scattering.

Introduction

Upon their development in 1962, biosensors have allowed a sensitive means of monitoring analytes by combining a biological component with a physicochemical detector component. With the development of fluorescent probes such as QDs, advances in imaging technology allow biosensors to report activities of specific targets and biomarkers [Morris 2010]. For example, enzymes can be used as biomarkers for the purpose to measure their activity which can help determine the rate of the reaction that they catalyze. There are many enzymes found within the human body, but a specific family of enzymes called matrix metalloproteinases (MMPs) are found to take part in a large number of physiological and pathological processes. Depending upon their expression, the activity of these enzymes greatly affects the balance between these types of processes. Because of this, it is of great interest to study these enzymes with the intent to figure out their mechanism of action so that it may become easier to combat diseases caused by over and under expression of these enzymes. It is important to note that the biological component of the biosensor will be the substrate that is part of the reaction catalyzed by the MMP.

As of now, a real-time in vivo biosensor that could be used to measure MMP activity has not been expanded upon or explored in literature papers. This may be caused by the fact that enzyme activity is very biologically dependent. This means that different conditions and various factors can have drastic effects on an enzyme's ability to catalyze a reaction. Such examples include temperature, pH, and other components that exist within the environment. Because of this, performing experiments under in vitro conditions, where lab environment is not the same as the local environment of the enzymes inside the body, may not lead to the true enzymatic activity that MMPs exhibit. In vitro measurements would lead to a lower the enzymatic activity due to a decrease in temperature [Melville 2011]. This is due to room temperature being about 25oC and the average body temperature of a human running around 37oC. The proposed in vivo biosensor will allow for the actual kinetic parameters of MMP activity to be measured without the use of multi-step assay processes used for in vitro biosensing.

Background

Matrix Metalloproteinases

MMPs make up a large family of highly homologous Zn2+ endopeptidases that can hydrolyze and cleave peptide bonds after binding to specific non-terminal amino acids in peptides with specific amino acid sequences. These peptides include collagenases, gelatinases, stromelysins, matrilysins, elastases, and membrane-type MMPs [Verma 2007]. Although more are believed to exist, only 23 different types of human MMPs have been identified. Due to their ability to cleave peptides, they are responsible for degradation of the constituents of extracellular matrix as well as maintaining a balance of tissue synthesis and degradation. They are normally expressed at a minimum due to their role in many physiological processes and are strictly controlled by endogenous MMP inhibitors (MMPIs) and tissue inhibitors of MMPs (TIMPs). Just like all of the processes in the human body, pathways must be regulated to achieve homeostasis. If this balance is broken, then serious injuries and diseases can occur. In the case of MMPs, over expression results in an imbalance between MMP and TIMP activity. This can ultimately lead to various pathological disorders.

As stated before, MMPs are involved in a large number of physiological processes such as wound healing, immune response, inflammation, nerve growth, ovulation, embryonic development, apoptosis, etc. However, their abnormal activity is linked to large number of pathological conditions, such as cancer, metastasis, arthritis, central nervous system (CNS) disorders (including Alzheimer’s disease, multiple sclerosis, Lou Gehrig’s disease, neuropathic pain, etc.), nephritis, liver fibrosis, as well as many others.

To even play a role in the processes described above, it is important to recognize how MMPs are activated. Initially, they found in their inactive zymogen form. It is only after a biological change occurs that they are converted into their active form. Their activation can be initiated by injury, inflammation, other proteinases, foreign bodies, and pathogens.

Like most zymogens, once their activation has been initiated, a change in their original configuration takes place and their active site becomes exposed. MMP's active site consist of a highly reactive zinc ion that is connected to a pro-peptide sequence by a sulfide bond. The sulfur element in the amino acid cysteine participates in this bond. It is only after this bond is broken will the MMP become active. Fig. 1 is a diagram that displays

Fig. 1. Illustration demonstrating the activation of an MMP-2 enzyme by the removal of the pro-peptide sequence using the means of reactive oxygen species as well as other proteinases (after [Kansasamy 2010]).

reactive oxygen species (ROS) and other proteinases cleaving the pro-peptide sequence to allow for MMP activation.

Because MMPs are found to participate in a large number of pathological conditions, the possible biosensing capability of their activity that they offer is very attractive because it could lead to new way of early detection of these conditions at specific sites in the body. A prime example would be to place one of these proposed biosensors at a pain signaling site to detect and objectively characterize neuropathic pain. By being able to detect pain, new insights about the causation of pain could be achieved. As it stands, existing pain treatments for peripheral neuropathy only reduce pain by 25-40% in about 7 of the 15 million patients treated for the condition in the US [Dworkin 2007]. This is because there is a poor understanding behind the mechanisms that cause nerve pain. [Dev 2010]. Just as with many other disorders and diseases, the understanding of mechanisms by which they are associated with can lead to improved treatments. In this particular case, the identification of pathophysiological mechanisms that contribute to pain can introduce new ways to attack the problem [Jensen 2009]. Due to their known involvement but unknown mechanism of action in peripheral neuropathy, MMPs have become great candidates of study.

Abnormal MMP expression in the CNS has been seen in several studies that measured the activity of MMPs in traumatic nerve injuries. All 23 MMPs are believed to take part in not only similar processes, but also different ones due to each MMP cleaving peptide bonds at different specific amino acid motifs within the peptide sequence. For example, MMP-9 exhibited immediate up-regulation and induced pain upon nerve injury, while MMP-2 was seen to induce pain in a delayed manner [Kawasaki 2008]. Another example is seen with MMP-1 and MMP-3. Their expression is directly correlated to the degree of disc herniation in the lumbar region [Zigouris 2011]. These studies were in vitro applications that were conducted by using multistep assay processes, such as zymography, enzyme-linked immunosorbent assay (ELISA), or immunohistochemical (IHC) staining. The results of these simple techniques lead to more of the same rationale mentioned earlier that calls for more intensive research on MMPs. The proposed biosensing method would be able to demonstrate these results at site specific pain-processing sites could alleviate some of the mysteries behind the mechanisms of neuropathic pain. This would allow for the capability of monitoring MMP activity in real time in the direct vicinity of nerve lesions. Although it is extremely important to objectively identify patients in need of treatment, there are no methods in doing so. Peripheral neuropathy identification might be able to be achieved by using a novel biomarker that results from the designation of non-neuronal signaling mechanisms.

Due to the response and releasing of proinflammatory cytokines and MMPs, chronic pathological pain is found to be caused by spinal cord glia [Dev 2010]. Spinal MMPs have not only been linked to neuroinflammation, but they have also been identified to be involved in pain associated with peripheral nerve damage. This is seen when the release of activated MMPs from various cells is signaled by the sciatic nerve. The signaling cascade begins when these nerve terminals of peripheral axons project to the spinal cord. Once again it becomes evident that analyzing MMP activity could provide guidance in the understanding of how acute pain slowly evolves into chronic, pathological pain. This link between the two and how their features can affect the transition from one to the other is still cloudy, but the chronic activation of glia cells in the CNS by MMPs offers a potential explanation for this transition.

Similar hypotheses can be made for other pathological disorders as well as for abnormal activity found to take place in physiological conditions. Because there are so many different

MMPs and so many different processes that they are involved in, attaching different QD probes on the same fiber for the proposed biosensor concept would allow for simultaneous observation of a number of different MMPs.

Quantum Dots as In Vivo Probes

Quantum dots (QDs) are nanoscale particles of semiconducting material that release an emission of photons. The smaller the size of the QD, the higher the frequency of light that is emitted. Their ability to maintain a bright and stable fluorescence under different stresses presented in complex environments makes them great candidates for in vivo experiments. [Vashist 2006]. With their stability also comes the ability to permit the target fluorescence signal to be more precisely recognized from the excitation light. This is due to the large Stokes shift that causes the emitted wavelength to be larger than the absorption wavelength. This also leads to a lower amount of energy to be released in the photon that is involved in the emission. [Gao 2003]. Their stability also allows to be able to resist photobleaching which can cause the QDs to lose their fluorescence by photochemical destruction. Another benefit of QDs is their large absorption coefficients. This allows for QDs to exhibit efficient probe excitation under in vivo conditions.

QDs are fluorophores that can efficiently bring about multiple wavelength colors in an emission spectra with having only one excitation source. This is believed to be caused by QDs having broad absorption lines combined with narrow emission ones. This is not normally seen in traditional fluorophores because they display reverse characteristics [Alivisatos 2005]. One important aspect to note is that commercial QDs are composed of CdSe/ZnS. This poses a problem with the proposed biosensing system due to Cd being very toxic. This does not make well for any in vivo applications, so the synthesis of Cd free QDs that do not display any cytotoxicity is critical. Another toxic threat could be the QD being released into the body because it is unknown as to not only how QDs will be metabolically broken down, but also if they will have any long term effects. This is where the optical fiber tip that the QDS will be tethered down to comes into play as it will prevent them from becoming liberated inside the body.

Research Objective

The end product of the proposed research is to develop an in vivo QD-based biosensor that demonstrates the ability to detect MMP activity. The idea is to be able to perform site-specific, real-time sensing with the ability to be minimally invasive in the testing of hospital patients. This type of application could possibly be used to test for early signs of cancer, peripheral neuropathy, exposure to ionizing radiation, and to monitor wound healing.

The subproject assigned to me was to help in the synthesis of the proposed Cd-free, Mn-Doped ZnSe/ZnS QDs. These are believed to not show sign of cytotoxicity, but it is still unproven. I was also assigned to help in the bioconjugation of the MMP-substrate (peptide motif) to the Mn-Doped ZnSe/ZnS QDs and to help carry out tests that will ultimately provide a proof of concept demonstration that the bioconjugation was successful.

Methodology

Biosensor Design

The biological component of the proposed biosensor would be the MMP substrates (peptides) bioconjugated to QDs. The detector component would consist of an optical fiber tethered to the bioconjugated compound by the use of a silica tip. As stated before, this tethering would inhibit the QDs from being released into the body while in vivo measurements of MMP activity are being generated. MMPs interacting and cleaving the peptides attached to the QDs are believed to also affect the fluorescence by either changing the wavelength in the emission of the QDs or the intensity. In order to quantitatively justify these changes a standard calibration curve must be constructed by collecting sensing data from known concentration of MMPs in solution

Because MMP-2 and MMP-9 have been heavily researched upon, this intended biosensing project will first emphasize the affects it will have on the substrates these enzymes interact with. Eventually this proposed research would like to expand and obtain the ability to measure the activities of all MMPs by using a single fiber tip. In general, MMPs recognize and bind to certain motifs located within the peptide. After the bind, they cleave the peptide bond through hydrolysis. In the case of MMP-9, the peptide motif that it recognizes is found to be Val-Val-Pro-Leu-Ser—Leu-Arg-Ser [Turk 2001]. The peptide bond that it hydrolyzes and therefore cleaves is found to be between the Ser and Leu amino acid residues. MMP-9 substrate sequences that contain this motif will be bioconjugated to the surface of QDs so that observable changes in the fluorescence of the QDs can be quantitatively measured before and after MMP-9 cleaves the substrate, giving its enzymatic activity.

After determining several different concentrations of MMP-2 and MMP-9 enzymes of known activities, immobilized QDs at the silica surface of optical fiber tips will be engulfed in the solution of these enzymes. While reactions are taking place, a sensing plot will be established in order to associate MMP-2 or MMP-9 activity with changes fluorescence. The fluorescence will measured by a spectrometer. As done with a standard curve, detector limits of the biosensor will need to be established by determining the minimum and maximum sensing capacity.

In order to immobilize The QDs onto the silica surface, a silane coupling reagent, 3-mercaptopropyltrimethoxysilane (MPTMS), will covalently bind to the metallic surface of the QD via the thiol functional group within the reagent. This interaction is due to affinity metal has for thiol molecules. To achieve silane covalently bonded to silica with the thiol molecules at the surface, a monolayer of the silane coupling reagent must be formed over hydroxyl-modified silica. This can happen when hydroxyl groups react with the hydrolyzable regions of MPTMS and form stable oxane bonds (Fig. 2). Immobilization of QDs that become covalently bonded to the thiol monolayer from the MPTMS happens because of the metal affinity displays for the thiol molecules (Fig. 3). The optical setup for the biosensor will consist of a diode laser excitation source coupled to a fiber, a Y-fiber (fiber coupler) for dual entrance and exit of the excitation and emission light, respectively, and a spectrometer with an array detector at the output end (Fig. 4).

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Fig. 2. Depiction of (a) a hydroxyl modified SiO2 surface and (b) a SiO2 surface modified with the silane coupling reagent 3-mercaptopropyltrimethoxysilane resulting in a monolayer of thiol functional groups covalently bonded to SiO2.

Fig. 3. Model of a QD tethered to the surface of silica due to covalent bonding from the silane coupling reagent.

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Fig. 4. Demonstration of biosensor setup that encompasses the implementation of QD-modified optical fibers to quantify MMP activity by exciting the QDs with light through one input of the fiber coupler. The light emission can then be measured by a detector array at the output of a spectrometer (not shown in the figure).

Direct Biosensing Concept

As aforementioned, QDs have highly sensitive surface properties that cause the ability of optical properties of the photoluminescence to change upon surface modification. With that being said, the proposed bioconjugation of substrates to QDs can drastically alter the QDs surface properties and ultimately lead to changes in their fluorescence emission because the substrates would be attached to the QDs on their surface. This change in fluorescence emission would be associated with the wavelength, so it would not be unexpected to see a change in wavelength due to a change fluorescence after a substrate has been cleaved from the QD. This change in QD fluorescence that can occur from the MMPs cleaving peptides is intended to be used and interpreted in a quantifiable analysis of MMP activity. Fig. 5 illustrates the idea of changes in fluorescence of QDs modified with peptides as a result of active MMPs cleaving the peptides. In Fig. 5a, a QD with the MMP-9 substrate conjugated to its surface emits orange fluorescence when excited by blue laser light and in Fig. 5b the same QD, under the same excitation source, emits green fluorescence after the peptide has been cleaved by an active MMP-9 enzyme.

Fig. 5. Illustration of QD fluorescence (a) before and (b) after the surface peptide is cleaved.

Description of Experiments

Synthesis and Characterization of High-Efficiency Cd-Free Colloidal Quantum Dots

The PI group that I have been working with on this project has performed a dubious amount of research in regards to nanoparticles, so the literature papers that they have published have allowed me access to all the infrastructure necessary to conduct the proposed research for the biosensor. This also includes everything from a laboratory for colloidal synthesis of nanoparticles (NPs) to the extensive characterization of tools needed. The research team is experienced in synthesis and characterization of a variety of II-VI [Greenberg 2007a, 2007b], [Osiński 2007], [Sankar 2007], [Akins 2010], [Osiński 2010b, 2011], [Akins 2012], III-V [Greenberg 2005, 2006a, 2006b], lead-halide [Sankar 2008], [Osiński 2009a], [Withers 2009b], lanthanide-halide [Withers 2008b, 2009a], [Sankar 2009], [Osiński 2009b, 2010a], [Plumley 2010], [Rivera 2011b, 2012], [Withers 2012] and lanthanide oxide [Rivera 2011a] semiconductor core and core/shell NPs, as well as superparamagnetic iron oxide NPs [McGill 2009a, 2009b], [Armijo 2012b], multifunctional iron oxide/II-VI NPs [Vargas 2011], [Armijo 2012a] and metallic core/semiconductor shell NPs [Kruse 2011].

Synthesis and Characterization of Mn-doped ZnSe/ZnS QDs

The CD-free QDs synthesized were Mn-doped ZnSe/ZnS. This choice was driven by not only their bright emission at room temperature and above [Pradhan 2005, 2007], [Akins 2012], but also because they share the same shell composition as in the standard CdSe/ZnS QDs. This is important because these commercial QDs allow for modification of previously developed conjugation techniques. Our preliminary experiments involving synthesis and optical measurements of Mn-doped ZnSe/ZnS colloidal QDs have revealed a notable change in the photoluminescence (PL) properties of ZnSe cores after subsequent hydrophilic ZnS shell

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synthesis, suggesting sensitivity of the QD emission to surface conditions.The colloidal synthesis of our ZnSe:Mn/ZnS QDs was adapted from [Pradhan 2005] and

[Acharya 2010] with substantial improvements. The ratio of Mn to Zn precursors, the large excess of Se at the beginning of synthesis, and the addition of S to the shell all account for the significant modifications to the synthesis procedure that have resulted in QDs with record high quantum efficiency (QE) exceeding 90% [Akins 2012]. In a typical synthesis, the Se precursor was prepared by adding 123 mg of selenourea to 10 mL of oleylamine in a three-neck flask. The solution was heated and stirred under argon until the selenourea was completely dissolved, then it was allowed to cool. The core Zn precursor was prepared by adding 63 mg of zinc stearate (ZnSt2) and 5 mL of octadecene (ODE) to a three-neck flask. The flask was then heated to 260 °C under argon; once the temperature was stabilized at 260 °C, all of the Se precursor was injected into the flask. The flask was then cooled to 240 °C. The Mn dopant was prepared by adding 6.3 mg of manganese stearate (MnSt2) to 0.5 mL of ODE. The mixture was injected into the flask at 240 °C, the temperature was maintained until the dopant emission appeared, and the flask was then heated to 260 °C.

The shell Zn precursor was prepared by adding 0.63 g of ZnSt2 to 0.28 g of stearic acid (SA) and 10 mL of ODE in a three-neck flask. The ZnSt2/SA/ODE solution was then heated and stirred under argon for injection. To begin, 0.96 mL of the ZnSt2/SA/ODE solution was injected into the flask at 260 °C, and after 10 min another 0.96 mL was injected into the flask. The remainder of the shell was grown by progressively increasing injections of the ZnSt2/SA/ODE solution into the flask. The injection temperature was 260 °C, and after each injection the temperature was rapidly lowered to 240 °C. The four subsequent injections had the volumes of 1.33 mL, 1.69 mL, 2.25 mL, and 2.81 mL, respectively.

The shell S precursor was prepared by adding 15 mg of powder S to 1.5 mL of ODE, which was then injected into the flask at 260 °C. The flask was cooled to 240 °C for 15 minutes, then cooled to room temperature. The resulting QDs were washed by centrifugation using acetone, until a transparent supernatant was obtained. The supernatant was then discarded, while the QD precipitate was collected using toluene and stored in a brown glass bottle.

Modifying Silica Surface with Mn-doped ZnSe/ZnS QDs

In order to covalently bond the synthesized Mn-doped ZnSe/ZnS QDs to a SiO2 surface for the intended application of optical fiber based MMP biosensing, a silane coupling agent, 3-mercaptopropyltrimethoxysilane (MPTMS), was used to create a thiol monolayer over SiO2

microscope slides. Due to the thiol metal affinity, it was observed that QDs could be covalently bonded to the thiol groups at the surface of the SiO2.

The slides were hydroxyl-modified based on a protocol outlined in [Metwalli 2006], where the slide was submerged in KOH solution for 24 hours, followed by sonication in water, submersion in an HCl solution, sonication with water again, and submersion in methanol. The slides were silanized based on a modification of a protocol outlined in [Hu 2001], where the hydroxyl-modified silica was submerged in 1% MPMTS in anhydrous benzene. Afterwards, the colloidal QDs were dropped onto the slide and mixed in a shaker.

Synthesis and Characterization of Bioconjugation of MMP-Substrate and Mn-doped ZnSe/ZnS QDs

This bioconjugation method was adapted from [Shi 2007] with improvements to avoid destroying the QD's fluorescence. The first step was to prepare the QD solution so that they could be extracted from the chloroform. One uM QDs in chloroform were centrifuged and dispersed in benzylamine and DMF. Then the peptides were solubilized in 200 uL of DMF so that they could be used in the bioconjugation process. This peptide solution was then added to the QD solution prepared earlier and was vortexed for 30 minutes. It was then subject to centrifugation, spin dialysis, and stored in a PBS solution. This was done with both pre-cleaved and cleaved peptides as to emulate the activity of MMP-9.

Preliminary Results

Preliminary Results on Synthesis and Characterization of Mn-doped ZnSe/ZnS QDs

PL spectra and QE were measured using a Horiba Jobin Yvon Fluorolog-3 spectrofluorometer, using Spectrosil® quartz cuvettes. The synthesized QDs exhibited multiple PL peaks that were not observed simultaneously but rather one emission line at a time, with each line being excited by a particular wavelength, which suggests different optical transitions. Figs. 6a and 6b are the PL excitation and emission spectra, respectively, for one of the transitions that take place for Mn-doped ZnSe/ZnS QDs dispersed in chloroform. The orange 597 nm emission excited by 400 nm wavelength shown in Fig. 6 is believed to be the result of interatomic transitions due to the Mn doping. The QE for these QDs due to the Mn doping was measured to be about 86.5%. The interesting thing to note is that when excited at 436 nm, two emissions are found to exist, one of which is believed to be some sort of surface defect emission. Fig. 7 displays that the emission believed to be caused by the surface defect exhibited a QE of 4.2%.

Fig. 6. Photoluminescence excitation (PLE) (a) and PL (b) spectra of Mn-doped ZnSe/ZnS QDs in toluene. For the PLE measurement, a parked emission wavelength of 596 nm was used, and for the PL measurement, an excitation wavelength of 400 nm was used.

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Following the hydrophilization procedure(modified after [Li 2008]), in which the thiol group of mercaptoacetic acid attaches itself to the ZnS shell, while the carboxyl group makes the QDs dispersable in water, the green 487 nm emission line disappears, suggesting that the defect emission is surface sensitive. Consequently, the hydrophillic QDs would only exhibit an orange emission peak at 595 nm, with a significant broadening of the excitation spectrum. Analogously, we anticipate that by attaching peptides to the QDs through a thiol-based bond, the green emission will be quenched or substantially weakened, and will be progressively recovered as a result of MMPs cleaving the peptides. The ratio of orange emission to that of recovered green emission will be the basis for the optical transduction mechanism that will measure the MMP activity using the QDs.

Surface silanization, i.e. coating the QDs with MPTMS, was explored based on a protocol of [Garcia 2010], and not only did the QDs stick to silica with their fluorescence still intact, additional observations about the PL were made. Figs. 8a and b show PLE and PL emission spectra, respectively, for MPTMS-coated Mn-doped ZnSe/ZnS QDs. Note the absence of the defect green emission that is generally seen in untethered QDs, further suggesting that this particular defect emission is surface sensitive.

Preliminary Results on Modifying Silica Surface with Mn-doped ZnSe/ZnS QDs

In order to verify that the QDs stayed attached as a result of thiol binding after washing the slide with toluene, the absorption properties of the glass slides were measured for the QD-

Fig. 7. PLE (a) and PL (b) spectra for Mn-doped ZnSe/ZnS QDs in chloroform, respectively. For the PLE measurements, (a) displays the excitation spectrum for ZnSe:Mn/ZnS QDs in chloroform using 487 nm parked emission wavelength. For the PL measurement, (b) displays the emission spectrum for ZnSe:Mn/ZnS QDs inchloroform using 436 nm excitation wavelength which gives rise to two different emissions of 487 and 595 nm.

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Fig. 8. PLE (a) and PL (b) spectra of MPTMS-modified Mn-doped ZnSe/ZnS QDs in toluene. For the PLE measurement, a parked emission wavelength of 588 nm was used, and for the PL measurement an excitation wavelength of 382 nm was used.

modified slide, a blank slide, and a control slide, which was not silanized but still had QDs dropped over its surface and mixed in the shaker. Fig. 9 shows the absorption spectrum for Mn-doped ZnSe/ZnS QDs in toluene, where an absorption feature can be observed at 423 nm. The absorption measurements taken from a blank slide and the QD-modified slide can be seen in Fig. 10a. The absorption feature at 423 nm can be observed for the QD-modified slide but not for the blank slide. Furthermore, in Fig. 10a, the 423 nm absorption feature can be observed more clearly in the absorption spectra for the QD-modified slide with the absorption line of the blank slide subtracted out. The results of absorption measurement for the control slide can be seen in Fig. 10b. The absorption feature at 423 nm observed for the QD-modified silica in Fig. 10a cannot be seen in Fig. 10b.

Preliminary Results of Synthesis and Characterization of Bioconjugation of MMP-Substrate and Mn-doped ZnSe/ZnS QDs

In a similar fashion to QE determination and the PL spectra shown above, the bioconjugation of the pre-cleaved and cleaved peptides to their respective QDs was measured and analyzed. It was determined that the wavelength of emission did not change much when comparing the two, but the QE interestingly enough did change. These observations can be seen in both Fig. 11 and 12. Fig 11 displays the spectra's for the QDs conjugated to the pre-cleaved peptides while Fig. 12 shows the spectra's for the QD's conjugated to the cleaved peptides. In both cases the optimal excitation was found to be around 320 nm, but that is not realistic for in vivo applications due to that falling in range ultra violet light in the electromagnetic spectrum. Instead, a 400 nm

Fig. 9. Absorption spectrum for Mn-doped ZnSe/ZnS QDs in toluene, showing an absorption feature at 423 nm.

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Fig. 10. (a) Absorption spectra lines for a blank SiO2 slide, a QD-modified SiO2 slide, and the QD-modified slide with the blank slide subtracted. Note the Mn-doped ZnSe absorption feature at 423 nm for the QD-modified slide. (b) Absorption spectra line for the control slide.

excitation was used when determining the QE for each. The pre-cleaved exhibited a QE of 39.1% while the cleaved showed a QE of 90.8%. In addition to these findings, it is important to note that the surface defect mentioned before disappeared on both spectra's, so the ratio comparison between the surface defect emission and the Mn-doped emission could not be further expanded upon.

Fig. 11. PLE (a) and PL (b) spectra for bioconjugated pre-cleaved peptides and Mn-doped ZnSe/ZnS QDs in PBS, respectively. For the PLE measurements, (a) displays the excitation spectrum using 596 nm parked emission wavelength. For the PL measurement, (b) displays the emission spectrum using 320 nm excitation wavelength.The QE was measured to be 39.1% by using a parked excitation wavelength of 400 nm.

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An additional test, dynamic light scattering (DLS), was run to verify the addition of the peptides to the QDs. DLS is used to determine the size distribution profile of small particles in suspension. It's used to measure the radius of the nanoparticle by giving an average distribution of size. In the comparison of the conjugated pre-cleaved peptides to QDs vs cleaved, it would be expected to see a decrease in radius after the peptide has been cleaved. This thought process turned out to be true as the pre-cleaved displayed a radius of 3.53 nm and the cleaved giving 3.26 nm as shown in Fig. 13a and b, respectively.

Conclusion

In conclusion to my subproject within the entire proposed research, it appears as though the bioconjugation of peptides to QDs was successful, although other ways to detect it should be used in order to further strengthen this belief. Additional bioconjugations need to be synthesized in order to prove that the project is repeatable. Although the PL spectra revealed that there was almost no change in wavelength after the peptide was cleaved, there was a change in intensity as seen in the increase in QE. This is still good news because it indicates that some change has occurred and leads to the belief that enzyme activity can still be measured by similar means.

Future Work

Fig. 12. PLE (a) and PL (b) spectra for bioconjugated cleaved peptides and Mn-doped ZnSe/ZnS QDs in PBS, respectively. For the PLE measurements, (a) displays the excitation spectrum using 597 nm parked emission wavelength. For the PL measurement, (b) displays the emission spectrum using 315 nm excitation wavelength.The QE was measured to be 90.8% by using a parked excitation wavelength of 400 nm.

Fig. 13. DLS for both pre-cleaved (a) and cleaved (b) peptides conjugated to QDs are depicted in the picture above. The pre-cleaved attached to the QDs were found to have an average of 3.53 nm while the cleaved displayed an average of 3.26 nm.

One major flaw within this proposed biosensor is that the Mn-Doped ZnSe/ZnS QDs have not been proven to be non-toxic. A cytotoxicity assay should be run to see if these QDs are safe for this in vivo application. As for the rest of the project, the development of a suitable biosensor should include the linking QDs to fiber tips, experiments with MMP activity detection, the construction of fiber-optic biosensor, and the calibration of fiber-optic biosensor

References

[Acharya 2010] S. Acharya, D. D. Sarma, N. R. Jana, and N. Pradhan, “An alternate route to high-quality ZnSe and Mn-doped ZnSe nanocrystals”, J. Phys. Chem. Lett. 1, pp. 485-488, 2010.

[Akins 2010] B. A. Akins, G. Medina, T. A. Memon, A. C. Rivera, G. A. Smolyakov, and M. Osiński, “Nanophosphors based on CdSe/ZnS and CdSe/SiO2 colloidal quantum dots for daylight-quality white LEDs”, Conf. Digest, 13th Annual Conf. on Lasers and Electro-Optics CLEO 2010, San Jose, CA, 16-21 May 2010, Paper CtuNN7.

[Akins 2012] B. A. Akins, A. C. Rivera, N. C. Cook, G. A. Smolyakov, and M. Osiński, “ZnSe:Mn/ZnS high temperature nanophosphors with very high quantum efficiency for white LEDs”, Techn. Digest, 32nd Annual Conf. on Lasers and Electro-Optics CLEO 2012, San Jose, CA, 1-6 May 2012, Paper CW1L.2.

[Alivisatos 2005] A. P. Alivisatos, W. Gu, and C. Larabell, “Quantum dots as cellular probes”, Annual. Rev. Biomed. Eng. 7, pp. 55-76, 2005.

[Armijo 2012a] L. M. Armijo, Y. I. Brandt, N. J. Withers, J. B. Plumley, N. C. Cook, A. C. Rivera, S. Yadav, G. A. Smolyakov, T. Monson, D. L. Huber, H. D. C. Smyth, and M. Osiński, “Multifunctional superparamagnetic nanocrystals for imaging and targeted drug delivery to the lung”, Colloidal Nanocrystals for Biomedical Applications VII (W. J. Parak, M. Osiński, and K. Yamamoto, Eds.), SPIE International Symposium on Biomedical Optics BiOS 2012, San Francisco, CA, 21-23 Jan. 2012, Proc. SPIE 8232, Paper 82320M (11 pp.).

[Armijo 2012b] L. M. Armijo, Y. I. Brandt, D. Mathew, S. Yadav, S. Maestas, A. C. Rivera, N. C. Cook, N. J. Withers, G. A. Smolyakov, N. L. Adolphi, T. C. Monson, D. L. Huber, H. D. C. Smyth, and M. Osiński, “Iron oxide nanocrystals for magnetic hyperthermia applications”, Nanomaterials 2 (#2), pp. 134-146, 7 May 2012.

[Dev 2010] R. Dev, P. K. Srivastava, J. P. Iyer, S. G. Dastidar, and A. Ray, “Therapeutic potential of matrix metalloproteinase inhibitors in neuropathic pain”, Expert Opin. Investig. Drugs 19 (#4), pp. 455-468, 2010.

[Dworkin 2007] R. H. Dworkin, A. B. O’Connor, M Backonja, J. T. Farrar, N. B. Finnerup, T. S. Jensen, E. A. Kalso, J. D. Loeser, C. Miakowski, T. J. Nurmikko, R. K. Portenoy, A. S. C. Rice, B. R. Stacey, R-D Treede, D. C. Turk, M. S. Wallace, “Pharmacologic management of neuropathic pain: Evidence-based recommendations”, Pain 132, pp. 237-251, 2007.

[Gao 2003] X. Gao and S. Nie, “Molecular profiling of single cells and tissue specimens with quantum dots”, Trends in Biotechnology 21 (#9), pp. 371-373, Sept. 2003.

[Garcia 2010] L. A. Garcia-Cerda, L. E. Romo-Mendoza, and M. A. Quevedo-Lopez, “Synthesis and characterization of NiO nanoparticles and their PMMA nanocomposites obtained by in situ bulk polymerization” J. Phys. Chem. Lett. 1, pp. 485-488, 2010.

[Greenberg 2007a] M. R. Greenberg, G. A. Smolyakov, T. J. Boyle, and M. Osiński, “Synthesis and characterization of ZnO and ZnO/ZnS nanocrystals”, Colloidal Quantum Dots for Biomedical Applications II (M. Osiński, T. M. Jovin, K. Yamamoto, Eds.), SPIE International Symp. on Biomedical Optics BiOS 2007, San Jose, CA, 20-21 Jan. 2007, Proc. SPIE 6448, Paper 644806 (11 pp.).

[Greenberg 2007b] M. R. Greenberg, G. A. Smolyakov, T. J. Boyle, and M. Osiński, “Synthesis and characterization of ZnO colloidal nanocrystals”, Techn. Digest, 27th Annual Conf. on Lasers and Electro-Optics CLEO 2007, Baltimore, MD, 6-11 May 2007, Paper CMO2.

[Hu 2001] M. Hu, S. Noda, T. Okubo, Y. Yamaguchi, and H. Komiyama, “Structure and morphology of self-assembled 3-mercaptopropyltrimethoxysilane layers on silicon oxide”, Applied Surface Science 181, pp. 307-316, 2001.

[Jensen 2009] T. S. Jenson, C. S. Madsen, and N. B. Finnerup, “Pharmacology and treatment of neuropathic pains”, Current Opinion in Neurology 22, pp. 467-474, 2009.

[Kawasaki 2008] Y. Kawasaki, Z. Xu, X. Wang, J. Y. Park, Z. Zhuang, P. Tan, Y. Gao, K. Roy, G. Corfas, E. H. Lo, and R. Ji, “Distinct roles of matrix metalloproteinases in the early- and late-phase development of neuropathic pain”, Nature Medicine 14 (#3), pp. 331-336, March 2008.

[Kruse 2011] D. A. Kruse, “Synthesis and Characterization of Core-Shell Nanomaterials for Solar Production of Hydrogen Fuel”, M.S. Thesis in Physics, University of New Mexico, April 2011.

[Li 2008] C. Li, M. Ando, H. Enomoto, and N. Murase, “Highly luminescent water-soluble InP/ZnS nanocrystals prepared via reactive phase transfer and photochemical processing”, J. Phys. Chem. 112, pp. 20190-20199, 2008.

[McGill 2009a] S. L. McGill, C. Cuylear, N. L. Adolphi, M. Osiński, and H. Smyth, “Enhanced drug transport through alginate biofilms using magnetic nanoparticles”, Colloidal Quantum Dots for Biomedical Applications IV (M. Osiński, T. M. Jovin, and K. Yamamoto, Eds.), San Jose, CA, 24-26 Jan. 2009, Proc. SPIE 7189, Paper 718918 (8 pp.).

[McGill 2009b] S. L. McGill, C. L. Cuylear, N. L. Adolphi, M. Osiński, and H. D. C. Smyth, “Magnetically responsive nanoparticles for drug delivery applications using low magnetic field strengths”, IEEE Trans. NanoBioscience 8 (#1), pp. 33-42, March 2009.

[Melville 2011] P. Melville, N. Benites, M. Ruz-Perez, and E. Yokoya, “Proteinase and phospholipase activities and development at different temperatures of yeasts of yeasts isolated from bovine milk”, J. Dairy Research 78, pp. 385-390, 2011.

[Metwalli 2006] E. Metwalli, D. Haines, O. Becker, S. Conzone, and C. G. Pantano, “Surface characterization of mono-, di-, and tri-aminosilane treated glass substrates”, J. Colloid and Interface Science 298, pp. 825-831, 2006.

[Osiński 2007] M. Osiński, K. Sankar, B. A. Akins, T. A. Memon, N. J. Withers, and G. A. Smolyakov, “Synthesis and characterization of ZnO/ZnS core/shell nanocrystals”, Invited Paper, Proc. 2007 US-Korea Conf. on Science, Technology, and Entrepreneurship (UKC2007), Global Challenges in Science and Technology, Symposium NST: Nano Science and Technology, Reston, VA, 9-12 Aug. 2007, Paper NST-7.2.

[Osiński 2009a] M. Osiński, “Colloidal synthesis and optical properties of lead-iodide-based nanocrystals (Invited Paper)”, Proc. International Conf. on Transport and Optical Properties of Nanomaterials (ICTOPON) (M. R. Singh and R. H. Lipson, Eds.), Allahabad, India, 5-8 Jan. 2009, AIP Conf. Proc. 1147, pp. 62-69.

[Osiński 2009b] M. Osiński, K. Sankar, N. J. Withers, J. B. Plumley, A. C. Rivera, B. A. Akins, and G. A. Smolyakov, “Lanthanide-halide-based nanoscintillators for portable radiological detectors (Invited Paper)”, Optics and Photonics in Global Homeland Security V and Biometric Technology for Human Identification VI (C. S. Halvorson, Š. O. Southern, B. V. K. V. Kumar, S. Prabhakar, and A. A. Ross, Eds.), SPIE Defense, Security, and Sensing Symp., Orlando, FL, 13-16 April 2009, Proc. SPIE 7306, Paper 730617 (16 pp.).

[Osiński 2010a] M. Osiński, J. B. Plumley, N. J. Withers, B. A. Akins, G. Medina, A. C. Rivera, and G. A. Smolyakov, “Synthesis and characterization of lanthanide halide scintillating nanocrystals for gamma radiation detection”, Proc. International Conf. on Nanoscience and Nanotechnology ICONN 2010 (A. Dzurak, Ed.), Sydney, Australia, 22-26 Feb. 2010, pp. 189-192.

[Osiński 2010b] M. Osiński, B. A. Akins, G. Medina, T. A. Memon, and G. A. Smolyakov, “Nanophosphors based on CdSe/ZnS colloidal quantum dots for daylight-quality white LEDs”, Proc. International Conf. on Nanoscience and Nanotechnology ICONN 2010 (A. Dzurak, Ed.), Sydney, Australia, 22-26 Feb. 2010, pp. 355-357.

[Osiński 2011] M. Osiński, B. A. Akins, J. B. Plumley, A. C. Rivera, N. C. Cook, A. Agarwal, and G. A. Smolyakov, “High-temperature nanophosphors for white-light-emitting diodes (Invited Paper)”, Proc. 16th Opto-Electronics and Communications Conf. OECC 2011, Kaohsiung, Taiwan, 4-8 July 2011 (4 pp.).

[Plumley 2010] J. B. Plumley, N. J. Withers, A. C. Rivera, B. A. Akins, J. M. Vargas, K. Carpenter, G. A. Smolyakov, R. D. Busch, and M. Osiński, “Thermal neutron detectors based on gadolinium-containing lanthanide-halide nanoscintillators”, Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XI (A. W. Fountain, III and P. J. Gardner, Eds.), SPIE Defense, Security, and Sensing Symp., Orlando, FL, 6-8 April 2010, Proc. SPIE 7665, Paper 76651F (13 pp.).

[Pradhan 2005] N. Pradhan, D. Goorskey, J. Thessing, and X. Peng, “An alternative of CdSe nanocrystal emitters: Pure and tunable impurity emissions in ZnSe nanocrystals”, J. Am. Chem. Soc. 127, pp. 17586-17587, 2005.

[Pradhan 2007] N. Pradhan and X. Peng, “Efficient and color-tunable Mn-doped ZnSe nanocrystal emitters: Control of optical performance via greener synthetic chemistry”, J. Am. Chem. Soc. 129, pp. 3339-3347, 2007.

[Rivera 2011a] A. C. Rivera, N. N. Glazener, N. C. Cook, B. A. Akins, J. B. Plumley, N. J. Withers, K. Carpenter, G. A. Smolyakov, R. D. Busch, and M. Osiński, “Detection of thermal neutrons using gadolinium-oxide-based nanocrystals”, Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XII (A. W. Fountain, III and P. J. Gardner, Eds.), SPIE Defense, Security, and Sensing Symposium, Orlando, FL, 25-29 April 2011, Proc. SPIE 8018, Paper 80180F (12 pp.).

[Rivera 2011b] A. C. Rivera, N. C. Cook, N. N. Glazener, N. J. Withers, J. B. Plumley, B. A. Akins, G. A. Smolyakov, R. D. Busch, and M. Osiński, “Dysprosium-containing nanocrystals for thermal neutron detection”, Nuclear Radiation Detection Materials (A. Burger, M. Fiederle, L. Franks, K. Lynn, D. L. Perry, and K. Yasuda, Eds.), 2011 MRS Spring Meeting, San Francisco, CA, 26-28 April 2011, MRS Symp. Proc. 1341, Paper U9.2 (6 pp.).

[Rivera 2012] A. C. Rivera, N. N. Glazener, N. C. Cook, S. Maestas, B. A. Akins, L. M. Armijo, J. B. Plumley, N. J. Withers, K. Carpenter, G. A. Smolyakov, R. D. Busch, and M. Osiński, “Thermal neutron detection with PMMA nanocomposites containing dysprosium fluoride

nanocrystals”, Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XIII (A. W. Fountain III, Ed.), SPIE Defense, Security, and Sensing Symp., Baltimore, MD, 23-27 April 2012, Proc. SPIE 8358, Paper 83581S (9 pp.).

[Sankar 2007] K. Sankar, B. A. Akins, T. A. Memon, S. T. Bowers, G. A. Smolyakov, and M. Osiński, “Colloidal synthesis of optically active ZnO/ZnS core/shell nanocrystals”, Symp. L: Zinc Oxide and Related Materials, 2007 MRS Fall Meeting, Boston, MA, 26-30 Nov. 2007, MRS Symp. Proc. 1035E, Paper L11.12 (available online).

[Sankar 2008] K. Sankar, B. A. Akins, T. A. Memon, N. J. Withers, G. A. Smolyakov, and M. Osiński, “Synthesis and characterization of scintillating lead-iodide-based nanocrystals”, Colloidal Quantum Dots for Biomedical Applications III (M. Osiński, T. M. Jovin, and K. Yamamoto, Eds.), San Jose, CA, 19-21 Jan. 2008, Proc. SPIE 6866, Paper 686604 (12 pp.).

[Sankar 2009] K. Sankar, J. B. Plumley, B. A. Akins, T. A. Memon, N. J. Withers, G. A. Smolyakov, and M. Osiński, “Synthesis and characterization of scintillating cerium-doped lanthanum fluoride nanocrystals”, Colloidal Quantum Dots for Biomedical Applications IV (M. Osiński, T. M. Jovin, and K. Yamamoto, Eds.), San Jose, CA, 24-26 Jan. 2009, Proc. SPIE 7189, Paper 718909 (12 pp.).

[Turk 2001] B. Turk, L. Huang, E. Piro, and L. Cantley, “Determination of protease cleavage site motifs using mixture-based oriented peptide libraries”, Nature Biotechnology 19, pp. 661-667, July 2001.

[Vargas 2011] J. M. Vargas, A. A. McBride, J. B. Plumley, Y. Fichou, T. A. Memon, V. Shah, N. Cook, B. A. Akins, A. C. Rivera, G. A. Smolyakov, J. R. O'Brien, N. L. Adolphi, H. D. C. Smyth, and M. Osiński, “Synthesis and characterization of core/shell Fe3O4/ZnSe fluorescent magnetic nanoparticles”, J. Appl. Phys. 109 (#7), Art. 07B536 (3 pp.), April 2011.

[Vashist 2006] S. K. Vashist, R. Tewari, R.P. Bajpai, L.M. Bharadwaj, and R. Raiteri, “Review of quantum dot technologies for cancer detection and treatment”, J. Nanotechnology online, 2006, http://www.azonano.com/Details.asp?ArticleID=1726.

[Verma 2007] R. P. Verma and C. Hansch, “Matrix metalloproteinases (MMPs): Chemical–biological functions and (Q)SARs”, Bioorganic & Medicinal Chemistry 15, pp. 2223-2268, 2007.

[Withers 2008] N. J. Withers, K. Sankar, T. A. Memon, B. A. Akins, J.-J. Gu, T.-Y. Gu, G. A. Smolyakov, and M. Osiński, “Fast-decay-time scintillation of LaF3:Ce colloidal nanocrystals”, Techn. Digest CD-ROM, 28th Annual Conf. on Lasers and Electro-Optics CLEO 2008, San Jose, CA, 5-9 May 2008, Paper CWM6.

[Withers 2009a] N. J. Withers, K. Sankar, J. B. Plumley, B. A. Akins, T. A. Memon, A. C. Rivera, G. A. Smolyakov, and M. Osiński, “Optimization of Ce content in Ce xLa1-xF3

colloidal nanocrystals for gamma radiation detection”, Nuclear Radiation Detection Materials – 2009 (D. L. Perry, A. Burger, L. Franks, K. Yasuda, and M. Fiederle, Eds.), 2009 MRS Spring Meeting, San Francisco, CA, 13-17 April 2009, MRS Symp. Proc. 1164, Paper L07-02.

[Withers 2009b] N. J. Withers, B. A. Akins, A. C. Rivera, J. B. Plumley, G. A. Smolyakov, and M. Osiński, “Lead-iodide based nanoscintillators for detection of ionizing radiation”, Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing X (A. W. Fountain, III and P. J. Gardner, Eds.), SPIE Defense, Security, and Sensing Symposium, Orlando, FL, 14-16 April 2009, Proc. SPIE 7304, Paper 73041N (12 pp.).

[Withers 2012] N. J. Withers, Y. I. Brandt, A. C. Rivera, N. C. Cook, L. M. Armijo, G. A. Smolyakov, and M. Osiński, “Effects of La0.2Ce0.6Eu0.2F3:Ce nanoparticles capped with polyethylene glycol on human astrocytoma cells in vitro”, Colloidal Nanocrystals for Biomedical Applications VII (W. J. Parak, M. Osiński, and K. Yamamoto, Eds.), SPIE International Symposium on Biomedical Optics BiOS 2012, San Francisco, CA, 21-23 Jan. 2012, Proc. SPIE 8232, Paper 82320R (9 pp.).

[Zigouris 2011] A. Zigouris, A. Batistatou, G. A. Alexiou, D. Pachatouridis, E. Mihos, D. Drosos, G. Fotakopoulos, M. Doukas, S. Voulgaris, and A. P. Kyritsis, “Correlation of matrix metalloproteinase-1 and -3 with patient age and grade of lumbar disc herniation”, J. Neurosurg. Spine 14, pp. 268-272, 2011.

[Shi 2007] L. Shi, N. Rosenzweig, and Z. Rosenzweig. "Luminescent Quantum Dots Fluorescence Resonance Energy Transfer-Based Probes for Enzymatic Activity and Enzyme Inhibitors", Analytical Chemistry. Vol. 79, No. 1, January 1, 2007: 208-214.

[Morris 2010] M. C. Morris. "Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporter to Modular Polypeptide Probes", Cell Biochem Biophys. DOI 10.1007/s12013-009-9070-7.