Throughout the experimental process of the project, we explored and evaluated numerous measurement methods. To present our choices and considerations, and to potentially assist future teams, we have created this page on measurement.

Choice for No Choice: Graphite Furnace Atomic Absorption Spectroscopy (GFAAS)

Throughout this project, the qualitative and quantitative analyses of various metal ions have been of great importance. Initially, we considered that inductively coupled plasma optical emission spectroscopy (ICP-OES) would be the most suitable method for high-throughput metal ion detection. However, given the high organic content present in our samples, ICP-OES without specialized treatment equipment poses significant challenges. Organic matter is difficult to completely combust in the plasma, leading to carbon buildup that can damage the instrument. As a result, we opted not to use ICP-OES for measuring our samples.

We further reviewed the national standard detection methods for the four elements: Li, Co, Ni, and Mn. Spectrophotometry (SP), Atomic Absorption Spectroscopy (AAS), and Energy Dispersive X-Ray Spectroscopy (EDX) are among the most commonly used and widely applicable techniques.

For Spectrophotometry (SP), the required colorimetric compounds differ for each element, leading to varying experimental methods. This approach is not suitable for complex systems containing multiple metal ions and organic acids; it requires stringent removal of impurities. To avoid overly complicated procedures and to minimize systematic errors arising from different experimental methods, we decided to forgo SP. Similarly, Energy Dispersive X-Ray Spectroscopy (EDX) was ruled out due to its inability to detect lithium (Li) and its high costs.

After extensive consideration, we ultimately selected Atomic Absorption Spectroscopy (AAS) as our final detection method. This technique allows for automated continuous sample introduction using a robotic arm, significantly reducing both labour and time costs. Additionally, for the same sample, we can easily switch between hollow cathode lamps for different elements, enabling the detection of all four target elements (Li, Co, Ni, and Mn) with greater convenience and efficiency.

We prepared standard solutions of the four metal ions and used them to generate calibration curves for each corresponding metal ion.

Fig. 1 Calibration Curves for the Detection of Four Elements via AAS

We utilized this calibration curve to determine the metal content in the black powder, comparing the obtained results with the data provided by the supplier. We have sufficient justification to accept this calibration curve. All metal ion detections in this project were conducted using Atomic Absorption Spectroscopy (AAS).

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High-Performance Liquid Chromatography (HPLC) is an advanced separation technique widely used across various fields, including chemistry, biology, pharmaceuticals, food science, and environmental science. In this project, it was applied to the quantitative analysis of organic small molecules such as citric acid and glucuronic acid.

Fig. 2 Calibration Curve for Citric Acid Detection via HPLC

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Evolutionary cycle for biomineralization—Scanning Electron Microscope-Energy Dispersive Spectrometer (SEM-EDS)

In the biomineralization module, we aimed to characterize the surface morphology of the microbial cells with the goal of visually observing the formed carbonate particles or improvements in the surface morphology of the cells.

Try-1 Optical Microscope

Fig. 3 Optical Microscopy Observations of Biomineralization Results. a) BL21(DE3) Control Group; b) Dual-Plasmid Experimental Group Li Mineralization; c) Dual-Plasmid Experimental Group Mn Mineralization; d) Dual-Plasmid Experimental Group Ni Mineralization; e) Dual-Plasmid Experimental Group Co Mineralization

We initially attempted to directly observe the surface of engineered Escherichia coli using an optical microscope. The observations indicated that, compared to the BL21(DE3) control group, the dual-plasmid experimental group expressing recombinant urease exhibited significantly different mineralization effects for the four metal ions (Li, Mn, Co, and Ni) under optical microscopy. Notably, the bacteria appeared to agglomerate, with a distinct layer of material adhering to their surfaces. However, it was challenging to obtain a clearer view of the specific details, prompting us to seek more suitable methods for further investigation.

Try-2 Confocal Microscope

To further investigate whether the material adhering to the bacteria in the experimental group corresponds to the respective carbonate mineralization products, we conducted comparative observations using a Confocal Microscope based on the recommendations of Professor Ma Yurong (see HP for details) . The results are as follows:

Fig. 4 Confocal Microscope Observations of Biomineralization Results. a) Control Group for Ca; b) Control Group for Li; c) Control Group for Mn; d) Control Group for Ni; e) Control Group for Co; f) Experimental Group for Ca; g) Experimental Group for Li; h) Experimental Group for Mn; i) Experimental Group for Ni; j) Experimental Group for Co

However, during the observations with the Confocal Microscope, we were unable to zoom in sufficiently to examine the surface details of individual bacteria. Although the experimental group exhibited more pronounced black shadows and aggregation phenomena compared to the control group under the same parameters, this still did not provide compelling evidence for our mineralization phenomenon.

Try-3 Polarizing Microscope

After consulting with Professor Ma again (see HP for details) , she recommended that we use a polarizing microscope for our observations. This is because carbonate minerals exhibit birefringence due to their unique crystal structures, which results in distinctive optical characteristics, such as interference colours and extinction positions. These features will assist us in determining whether the surfaces of our mineralization products are coated with carbonate minerals.

Fig. 5 Polarizing Microscope Observations of Biomineralization Results (Left: Dark Stripes; Right: Light Stripes)

Calcium carbonate crystals appear white under dark stripes and black under light stripes in a polarizing microscope. However, our biomineralization samples did not exhibit crystal birefringence under the dark stripes. Subsequently, we directly observed nickel carbonate and cobalt carbonate and found that the carbonate precipitates also did not exhibit crystalline properties under the polarizing microscope. Upon consulting with Long Haitao, who manages the polarizing microscope, we learned that some crystals (such as silicon dioxide) may not display the expected luminescent phenomena in dark-field observations under the polarizing microscope. This suggests that the polarizing microscope may not effectively illustrate our mineralization results. Furthermore, it is possible that the requirements of the polarizing microscope for samples to be transparent or semi-transparent were not met by our biomineralization samples.

Try-4 Atomic Force Microscopy(AFM)

During our literature review, we learned that Atomic Force Microscopy (AFM) has become an ideal tool for observing biomineralization results due to its high resolution, three-dimensional imaging capabilities, and non-destructive nature. AFM can reveal structural details of biominerals at the nanoscale, measure mechanical properties, and monitor the mineralization process in real time, providing intuitive and precise data to enhance our understanding of the mechanisms underlying biomineralization. Therefore, we will next attempt to analyze the surface morphology and height of the bacteria using AFM to assess our mineralization results. (Note: We will initially conduct AFM observations and analyses on the mineralization of the dual-plasmid experimental group for Ca and the control group to preliminarily evaluate the feasibility of AFM for mineralization analysis. Details of AFM analyses for Li, Mn, Co, and Ni can be found in the results section).

Fig. 6 AFM Observations of Biomineralization Results. a) Dual-Plasmid Experimental Group for Ca; b) BL21 Control Group

It can be observed that the bacteria in the control group are relatively dispersed, with a flatter surrounding area. In contrast, the dual-plasmid experimental group exhibits noticeable aggregation of bacteria, with height differences evident around them, confirming the presence of some adhering material. However, due to the limitations of the AFM instrument, it is not effective for observing bacterial structures at scales reaching several tens of micrometers and height discrepancies of several micrometers, as this may damage the probe.

Try-5 Scanning Electron Microscope-Energy Dispersive Spectrometer(SEM-EDS)

Based on literature reviews and the guidance of our supervisor, we attempted to use Scanning Electron Microscopy (SEM) to observe the results.

Since calcium carbonate biomineralization is the most common, we used the biomineralization of Ca²⁺ as an example to verify the mineralization level of the recombinant urease. Details of the SEM and Energy Dispersive Spectroscopy (EDS) analyses for Li, Mn, Co, and Ni can be found in the results section (due to the limitations of the scanning electron microscope, elements below carbon cannot be analyzed by EDS; hence, Li cannot be analyzed using EDS).

Fig. 7 SEM Images of Ca²⁺ Biomineralization Results. a) SEM Images of Ca negative control; b) SEM Images of the Ca experimental group; c) SEM Images of Ca positive control

Fig. 8EDS analysis of Ca. a)Schematic diagram of Ca carbonate biomineralization; b)EDS layered images of Ca; c)EDS analysis of Ca negative control; d)EDS analysis of the Ca experimental group; e)EDS analysis of Ca positive control

As shown in the figure, there are clear distinctions between the Ca²⁺ experimental group and the positive control group. The bacteria in the experimental group exhibit swelling in certain areas, forming a club-like shape, while the positive control group shows hemispherical carbonate precipitates surrounding the bacteria, with more pronounced intercellular adhesion. In contrast, the negative control bacteria have relatively smooth surfaces with no signs of aggregation.

Additionally, we utilized the elemental analysis feature of EDS to observe in situ what elements the rough protrusions correspond to. The EDS results indicated that the calcium (Ca) content analysis follows the trend: positive control > experimental group > negative control. The results were consistent and favorable, leading us to ultimately select SEM-EDS as the final characterization method for the mineralization portion of our study.

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Morphological Characterization of the Assembled Body—Atomic Force Microscope (AFM)

In the morphological characterization of peptide self-assemblies, we chose to use Atomic Force Microscopy (AFM) to observe and characterize the morphology of the assemblies, given that the average particle size and height differences are primarily at the nanoscale. Compared to the bacteria, which have significant height variations and reach micrometer scales, AFM demonstrated better observational capabilities. The results are as follows:

Fig. 9 AFM Analysis Schematic

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