Connection to the Integrative Project

Quantum dots (QDs) and nanorods (NRs), as semiconductor nanocrystals, are of fundamental and technical importance. Owing to their extraordinary optical properties and high quantum-yield efficiency, these nanoobjects are often geared towards many energy-relevant applications. In our IGEM project, we conceive to harness those nanoscale objects as solar energy harvester. When firmly anchored onto E. coli biofilms through coordination chemistry, they can be easily recycled together with scalable biofilm coatings when necessary, and meanwhile, still possess the capability to efficiently convert photons into electrons upon light exposure. The aquired electrons would then tap into the electron chains of engineered strain harboring hydrogenase gene cluster, thereby assisting the enzymes to fulfill hydrogen production.

Introduction and Motivation

Quantum dots (QDs) and nanorods (NRs) are semiconductor nanocrystals that have been widely studied and believed to be promising for many applications. Their unique electronic and optical properties arising from their unique quantum confinement effect and tunable attributes enable them to become versatile imaging tools and highly effective light harvesters. In our project, we conceive to harness those nanoscale objects as light hunter so that they can convert photons into transferrable electrons in our bio-abiotic hybrid system. In addition, we have utilized QDs as an imaging tool to visualize biofilms formation in our IGEM project.

When shed by light, QDs and NRs would capture photons with high energy, resulting in the excitation of electrons from low energy levels to higher ones. Without mediators, the unstable electrons would perform spontaneous transition to low energy levels. This electron transition would result in photoluminescence phenomenon and emit photons at specific wavelengths. On the other hand, if in the presence of electron mediators such as enzyme, the electron with high energy would transfer to the enzyme and participate in the catalysis reactions [1], this fact has originally inspired us to utilize QDs and NRs to assist hydrogenases to drive hydrogen production.

Figure 1. Different types of QDs with varied size and chemistry show different colors under UV light

Besides the difference in energy band structure and other chemical attributes, we take great interest in the difference in the shapes of the nanocrystals. After thorough literature research, we found that the merits of NRs, a one-dimension nanomaterial, may overweigh QDs’, a zero-dimension nanomaterial, in biohybrid catalysis in terms of their potential to induce conformational changes of enzymes and facilitate their biological functions if the shape fits. As suggested by a recent publication[1], the CdS NRs may contribute to N₂ production by inducing the conformational changes of nitrogenase. Therefore, we utilize a simple nanorods, CdS NRs and a typical quantum dots, CdSe QDs, for the comparison of catalysis efficiency in our projects.

Ligand is also an important feature of nanocrystals. The presence of some types of ligands would increase the difficulty of growth on some lattice plane while decrease it on the others, leading to NRs using QDs as nuclei. A ligand not only can serve as a stabilizer in solution but also a functional decoration for nanocrystals. In our experiements, the product QDs and NRs are originally synthesized with lipophilic ligands and dissolve in nonpolar organic solvents such as octadecene and trioctylphophine. Ligand exchanging would allow the products to enter into water phase and thereby enable ligand-decorated QDs that would be able to bind to biofilms. The ligand we used has a NTA functional group, which was developed by the Zhong group at ShanghaiTech (Patent application submitted). NTA-capped QDs or NRs can firmly bind with His-tagged CsgA, major protein components of E. coli biofilms. In addition, we further utilize this specific binding system and unique optical properties of QDs to scrutinize the expression and formation of biofilms (as revealed in our biofilms

session).

Key Achievements

Briefly, we achieved following goals:

1.Synthesized NTA-capped CdSe QDs and CdS NRs

2.Characterized the attributes of QDs and NRs with:(1)PL Spectrometer(2)UV-Vis Spectrometer(3)TEM

3.Nanomaterials with expected features ready for biofilms binding and for hydrogen production.

4.Nanomaterials for imaging and detecting biofilms growth and formation

Synthesis and Characterization

Figure 2. The flow chart for synthesis and characterization of nanomaterials.

Methods

Ligand Synthesis and Exchange:

To increase the solubility and enable selective binding ability of the nanomaterials, the lipophilic ligands of our synthesized nanomaterials should be replaced by special ligands through ligand exchange. We used a well-developed procedure (developed in Zhong Lab@ShanghaiTech) to synthesize this ligand. We performed the ligand exchange experiment to replace the origin ligand of the nanomaterials. The result of ligand exchange experiment was shown by the following image. After this experiment, the nanomaterials were ready to be harnessed as the binding material of the biofilm and the solar energy harvester of the Solar Hunter.

Ultraviolet-visible (UV-Vis) spectroscopy:

The Ultraviolet-visible (UV-Vis) spectroscopy is an absorption spectroscopy in the near-UV and visible spectral region. The UV-Vis spectroscopy is normally used as a quantitative and qualitative characterization for different analytes. As QDs and NRs solutions contain large amount of transition metal irons, they exhibit absorption in UV-Vis range due to stimulated transision. And after determined the molar absorptivity(ε) from the concentration of Cd²⁺, the molarity can be calculated by the following equation:

A=εBC

In the equation, A(Abs) refers to absorption; ε(Abs*cm^-1*M^-1) refers to molar absorptivity; B(cm) refers to the length of cuvette; C(M) refers to the molarity of the QD or NR solution.The UV-Vis spectrometer we use is Agilent Cary 5000.

Photoluminescence(PL) spectroscopy:

The Photoluminescence(PL) spectroscopy is a widely used tool for characterization of electronic and optical properties of analytes, especially quantum dots and nanorods. Briefly, the PL spectroscopy detects photon emitted in a certain spectral region when the samples are excited by light at a certain wavelength. The PL spectroscopy can be used to determine the excitation peak and emission peak. The position of the emission peak can indicate whether the synthesis of QDs and NRs is successfully performed. The best excitation wavelength and the best emission wavelength, which are of great importance for utilizing the use of QDs and NRs as characterization tools, can also be known from the position of the peaks. The PL spectrometer we use is HORIBA FL-3.

Transmission Electron Microscope(TEM):

Transmission Electron Microscope (TEM) was used to image the shapes and sizes of QDs and NRs. The 120KV TEM (National protein facility center, Shanghai, China) we used provides the resolution of around 0.2nm. The TEM sample of our lipophilic QDs and NRs solution is prepared by simply dropping 10 μL of the solution on to the copper grid.

Results

Synthesis and Characterization of CdS Nanorods

We synthesized CdS nanorods following a procedure adapted from a previously published protocol[1]. The synthesis procedure mainly contains two steps: synthesis of CdS seeds, followed by growth of CdS nanorods using CdS nanoparticles as nuclei.

Figure 3. Solutions of CdS nanoparticle seeds in TOP (left), CdS NRs in toluene (right)

Characterization of UV-Vis was performed to calculate the concentration of CdS seed in TOP solution and CdS nanorod in toluene solution. Also, PL spectrum of CdS NRs in toluene was collected to investigate the emission attribute of the nanorod. TEM image of the nanorods was acquired to study the shape and size distribution.

Figure 4. Result of CdS NRs ligand exchange experiments.

A ligand exchange experiment was performed and the result is shown in Figure 4

Figure 5. UV-Vis spectra of CdS seeds in TOP (A) and CdS nanorods in toluene (B);. Photoluminescence Spectrum of CdS nanorods in toluene (C).

The concentration of CdS seeds and CdS NR products were determined by using the UV-Vis spectrometer (Fig. 5 A, B). The peak shown in PL spectrum (Fig. 5 C) matches the absorption peak of the UV-Vis spectra, which thus proves the synthesis of CdS NRs.

Figure 6. TEM images of CdS NRs (A) and size distribution of CdS NRs (B). Note: 100 NRs in total were measured to determine the size distribution. The NRs thus measured have an average diameter of 3.93±0.57nm and average length of 66.81±6.74nm.

TEM confirms that synthesized products show nanorod feature, with an average diameter of 3.93±0.57nm and average length of 66.81±6.74nm.

Synthesis and Characterization of CdSe QDs

We followed a protocol from a previously published paper[2]. After synthesis, UV-Vis spectroscopy, PL spectroscopy and TEM were carried out to confirm the structures and features of CdSe QDs. The photoluminescence of CdSe QDs can be observed by naked eyes.

Figure 7. Experimental set-up for synthesis of CdSe QDs (a) , synthesized NTA-capped CdSe in PBS solution (b) and PL of CdSe QDs in PBS under UV light (c, excitation wavelength 354 nm)

A ligand exchange experiment was performed and the result is shown in Figure 8.

Figure 8. UV-Vis spectrum of CdSe QDs in CH₂Cl₂ (a); PL spectrum of CdSe QDs in CH₂Cl₂ (b)and The TEM image of CdSe QDs(c).

We used UV-Vis spectrum to confirm the successful synthesis of CdSe QDs. The absorption attribute of the QDs has indeed been approved (Fig 8. A). The QDs/CH₂Cl₂ exhibits an emission peak at 528nm, which matches the absorption peak of the UV-Vis spectra (Fig 8. B). Finally, we used TEM to show that CdSe QDs have nanoparticle features, with an average diameter of 6-8 nm (Fig 8. C).

Reference

1. K. A. Brown et al., Light-driven dinitrogen reduction catalyzed by a CdS: nitrogenase MoFe protein biohybrid. Science 352, 448-450 (2016).

2. C. Pu et al., Highly reactive, flexible yet green Se precursor for metal selenide nanocrystals: Se-octadecene suspension (Se-SUS). Nano research 6, 652-670 (2013).

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 </center>
 <h3 class="bg">Methods</h3>
-<p><b>Ligand Synthesis and Exchange:</b></p><p>
+<p><b class="bg1">Ligand Synthesis and Exchange:</b></p><p>
 To increase the solubility and enable selective binding ability of the nanomaterials, the lipophilic ligands of our synthesized nanomaterials should be replaced by special ligands through ligand exchange. We used a well-developed procedure (developed in Zhong Lab@ShanghaiTech) to synthesize this ligand. We performed the ligand exchange experiment to replace the origin ligand of the nanomaterials. The result of ligand exchange experiment was shown by the following image. After this experiment, the nanomaterials were ready to be harnessed as the binding material of the biofilm and the solar energy harvester of the Solar Hunter.</p><p></p>
-<p><b>Ultraviolet-visible (UV-Vis) spectroscopy:</b></p><p>
+<p><bv>Ultraviolet-visible (UV-Vis) spectroscopy:</b></p><p>
 The Ultraviolet-visible (UV-Vis) spectroscopy is an absorption spectroscopy in the near-UV and visible spectral region. The UV-Vis spectroscopy is normally used as a quantitative and qualitative characterization for different analytes. As QDs and NRs solutions contain large amount of transition metal irons, they exhibit absorption in UV-Vis range due to stimulated transision. And after determined the molar absorptivity(ε) from the concentration of Cd<sup>2+</sup>, the molarity can be calculated by the following equation: </p><center><p></p><p>A=εBC</p></center><p></p><p>In the equation, A(Abs) refers to absorption; ε(Abs*cm<sup>-1</sup>*M<sup>-1</sup>) refers to molar absorptivity; B(cm) refers to the length of cuvette; C(M) refers to the molarity of the QD or NR solution.The UV-Vis spectrometer we use is Agilent Cary 5000.</p><p></p>
-<p><b>Photoluminescence(PL) spectroscopy:</b></p><p>
+<p><b class="bg1">Photoluminescence(PL) spectroscopy:</b></p><p>
 The Photoluminescence(PL) spectroscopy is a widely used tool for characterization of electronic and optical properties of analytes, especially quantum dots and nanorods. Briefly, the PL spectroscopy detects photon emitted in a certain spectral region when the samples are excited by light at a certain wavelength. The PL spectroscopy can be used to determine the excitation peak and emission peak. The position of the emission peak can indicate whether the synthesis of QDs and NRs is successfully performed. The best excitation wavelength and the best emission wavelength, which are of great importance for utilizing the use of QDs and NRs as characterization tools, can also be known from the position of the peaks. The PL spectrometer we use is HORIBA FL-3.</p><p></p>
-<p><b>Transmission Electron Microscope(TEM):</b></p><p>
+<p><b class="bg1">Transmission Electron Microscope(TEM):</b></p><p>
 Transmission Electron Microscope (TEM) was used to image the shapes and sizes of QDs and NRs. The 120KV TEM (National protein facility center, Shanghai, China) we used provides the resolution of around 0.2nm. The TEM sample of our lipophilic QDs and NRs solution is prepared by simply dropping 10 μL of the solution on to the copper grid.</p><p></p>
 <h3 class="bg">Results</h3>
-<p><b>Synthesis and Characterization of CdS Nanorods</b></p><p>
+<p><b class="bg2">Synthesis and Characterization of CdS Nanorods</b></p><p>
 We synthesized CdS nanorods following a procedure adapted from a previously published protocol[1]. The synthesis procedure mainly contains two steps: synthesis of CdS seeds, followed by growth of CdS nanorods using CdS nanoparticles as nuclei. </p><p></p>
 <p></p>
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 </p>
-<p><b>Synthesis and Characterization of CdSe QDs</b></p><p>
+<p><b class="bg2">Synthesis and Characterization of CdSe QDs</b></p><p>
 </p><p><p></p>
 We followed a protocol from a previously published paper[2]. After synthesis, UV-Vis spectroscopy, PL spectroscopy and TEM were carried out to confirm the structures and features of CdSe QDs. The photoluminescence of CdSe QDs can be observed by naked eyes.</p>

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