You will find here a brief overview of some of the projects I have been involved in. I selected the ones I consider the most important and interesting, which allowed me to develop a higher understanding of physical phenomenon and technological development.

Controlled Translocation

The advent of analytical techniques with extremely low limits of detection led to dramatic progresses in the field of nucleic acids sequencing. I collaborated within the FET Open PROSEQO project, where we built upon current state-of-the-art sequencing technologies to develop novel proof-of-principle technologies for high-throughput protein sequencing and single molecule DNA/RNA sequencing.

This huge project has been divided in sub-tasks:

• a new sequencing technology that utilizes plasmonics to enhance the optical detection and to control the molecules movement by electrical trapping;
• a novel approach of plasmonic enhanced FRET and SERS spectroscopy for sequencing of proteins;
• a rigorous analytical model to reconstruct the exact sequence from the signals recorded;
• a single molecule sequencing device that can perform sequencing of both nucleic acids and amino-acids in one functional unit.

My duty for this project was to scale down to the micro/nano scale the electrophoresis technique, in order to study the possibility to control the single particle positioning with nanometer precision and drive selectively the particles into nano-structures. The nanometer precision on the particle positioning dramatically increases the single molecule detection, which is critical for the success of the project.

I created a microfluidic chamber to study the behavior of quantum dots in viscous mediums under an electrical field. I was interested in exploring the possibility to control the particle displacement with a nanometer precision, using the viscosity of the medium and the intensity of the electrical field as main variable parameters.

Concerning the experimental design, a PDMS box was filled with a mixture of gel and quantum dot. Two electrodes were placed in the box, and the tracking was performed with a CCD camera coupled with a standard upward microscope.

I created my own code to track the particle motion starting from the raw experimental recordings. A standard experimental recording is about 1 min with 60 fps. The resolution of my system was about 100 nm, but the goal of the post process analysis performed with my software was to reduce this number of one order of magnitude.

The first step of my code was to use the data to graphically represent the particle motion with a tracked path (a). Then the path was fitted with a line, to find the electrical field direction. A rough estimate of the particle velocity was done based on the time and the distance between the first and the last position. The calculate velocity was used to evaluate the average number of frames covered to overtake the optical resolution of the system. These information was used to create a new plot with only the positions compatible with the optical resolution of the microscope, and the data were rotated to represents the electrical field horizontally (b).

At this point, the motion along and orthogonal the electrical field were studied separately. Plotting the total displacement orthogonal to the electrical field it is clear that the particle is affected only by the Brownian motion. From the displacement along the EM field the speed of the particle in this viscous medium can be evaluated (about 0.1 um/sec) (a). Then,the frame-by-frame particle motions along and orthogonal the EM field (b), the frame-by-frame global motion (c) and the global motion with an average on 50 frame at a time were observed.

To better understand what happened in each frame and to see how precise we can be in controlling and tracking the particles, I created the histogram of both the orthogonal and horizontal frame-by-frame plot, and the histogram was fitted with a gaussian function. With this analysis we can extract the average motion of the particle during each frame. In the plot a) a average motion of 2 nm along the field is presented, and in b) 0 means orthogonal the field. After the analysis, the resolution can be improved of 2 order of magnitude from the instrumental resolution.

To conclude our understanding of the behavior of a single particle motion within a highly viscous medium under an EM field, I compared the motion of QD under several different EM conditions. The electric field was turned on with pulses of different lengths and frequencies keeping both the on/off ratio and the electrical potential constant, starting from the 1 ms pulse with 1 Hz to arrive to 10 ns pulse with 10 MHz at 10 V. The result is clear, showing no difference and apparently no threshold in the frequency: the particle has a minimum speed and it cannot displaced with a resolution below the one presented here.

The same experiment has been repeated without the electrical field.

All the plots and calculus presented here are performed by a single software I created with C++.

Intracellular Recording

The ability to accurately monitor electrogenic cells plays a pivotal role in neuroscience, cardiology and cell biology. Despite pioneering research and long-lasting efforts, the existing methods for intracellular recording of action potentials on the large network scale suffer clear limitations that prevent their widespread use.

I collaborated within a project where we showed that the synergistic combination of a planar porous electrode and plasmonic optoacoustic poration allows commercial CMOS-MEAs (CMOS-multi-electrode arrays) to record high-quality intracellular action potentials in large networks, performing optoporation and intracellular recording on all the electrod at the same time. The method is reliably used for both primary and immortalized cell types with the possibility of non-invasively testing a variety of relevant drugs.

My role in this project was to create a software able to handle the recorded data from all the electrodes of a CMOS-MEA (about 4000), post-process the raw data (about 7 million points per electrode) and performing the statistical analysis. I created a software that can perform several tasks on all the points of all the electrodes simultaneously.

The first issue was represented by the raw data, because the presence of several amplifier offsets throughout the recording was translated in several vertical shifts which hid the signal (a). The software recognizes all the shifts and creates a continues signal (b).

The resulting data present a time dependent baseline which prevents any kind of analysis of the signal (a). I created a baseline filter calculator to align the baseline in order to produce a readable signal (b).

At this point, the software recognizes the intracellular and extracellular spikes, saves each of them separately and estimates an upper limit of the Full Width Half Maximum (FWHM). The time dependence of the FWHM and height of the spikes are plotted with its histogram.

I studied the standard shape of an intracellular and extracellular signal and I evaluate a global function able to describe any cellular spike.

With this function, the software is able to fit all the spikes and to recognize the extracellular component into the intracellular signal when it appears. Once the signal is fitted, the FWHM and the height of any spike is evaluated precisely with the equation.

Then the time dependence of FWHM and the Height is presented, comparing the estimated value with the fitted one.

Because this study is performed on the entire multi electrode array, it is possible to compare the results throughout all the electrodes. It is also possible to investigate global patterns and behaviors on the entire cell culture. For example, I implemented a feature which produces the histogram of the FWHM and height of all spikes for each electrode getting the average value of FWHM and height on the entire experiment time. With this values it is possible to produce a new histogram representing the entire cell culture FWHM and height during the entire experiment.

The meaning of our results are discussed in our publication Intracellular Recordings on High-Density CMOS-MEAs by Plasmonic Meta-Electrodes, recently accepted on Nature Nanotechnology.