Andrea Cavallini

Multi-panel drugs detection in human serum for personalized therapy

This work focuses on P450 biosensors based on multiwalled carbon nanotubes (MWCNT) and different cytochrome isoforms: 3A4, 2B4, 2C9. The proposed biosensors exhibit enhanced sensitivities and decreased detection limits thanks to carbon nanotubes. The MWCNT structuring improves the sensitivity from 5.1 to 20.5 nA/mM mm(2) in case of CYP2B4-mediated Benzphetamine detection, from 0.26 to 0.63 nA/μM mm(2) in case of CYP3A4-mediated Cyclophosphamide detection, and from 0.11 to 0.25 nA/μM mm(2) in case of CYP2C9-mediated Naproxen detection. By using MWCNT, the limit of detection was enhanced from 59 to 12 μM in case of Cyclophosphamide and from to 187 to 82 μM in case of Naproxen. This makes possible the drug detection in human serum within the pharmacological range. In the paper, a new mathematical model is also proposed to succeed in discriminating different drug contributions in a mixture containing both Cyclophosphamide and Dextromethorphan or combining Naproxen and Flurbiprofen. Data analysis shows variations in reduction peaks that are dependent on the drug ratio, and that are consistent with competitive kinetics of substrates. This new approach enables multiple drug detection and opens the way to possible applications in personalized therapy.

Fully Integrated Biochip Platforms for Advanced Healthcare

Recent advances in microelectronics and biosensors are enabling developments of innovative biochips for advanced healthcare by providing fully integrated platforms for continuous monitoring of a large set of human disease biomarkers. Continuous monitoring of several human metabolites can be addressed by using fully integrated and minimally invasive devices located in the sub-cutis, typically in the peritoneal region. This extends the techniques of continuous monitoring of glucose currently being pursued with diabetic patients. However, several issues have to be considered in order to succeed in developing fully integrated and minimally invasive implantable devices. These innovative devices require a high-degree of integration, minimal invasive surgery, long-term biocompatibility, security and privacy in data transmission, high reliability, high reproducibility, high specificity, low detection limit and high sensitivity. Recent advances in the field have already proposed possible solutions for several of these issues. The aim of the present paper is to present a broad spectrum of recent results and to propose future directions of development in order to obtain fully implantable systems for the continuous monitoring of the human metabolism in advanced healthcare applications.

Continuous monitoring of Naproxen by a cytochrome P450-based electrochemical sensor

This paper reports the characterization of an electrochemical biosensor for the continuous monitoring of Naproxen based on cytochrome P450. The electrochemical biosensor is based on the drop-casting of multi-walled carbon-nanotubes (MWCNTs) and microsomal cytochrome P4501A2 (msCYP1A2) on a graphite screen-printed electrode (SPE). The proposed biosensor was employed to monitor Naproxen (NAP), a well-known anti-inflammatory compound, through cyclic voltammetry. The dynamic linear range for the amperometric detection of NAP had an upper limit of 300 µM with a corresponding limit of detection (LOD) of 16 ± 1 µM (S/N=3), which is included in NAP physiological range (9-300 µM). The MWCNT/msCYP1A2-SPE sensor was also calibrated for NAP detection in mouse serum that was previously extracted from mice, showing a slightly higher LOD (33 ± 18 µM). The stability of the msCYP1A2-based biosensor was assessed by longtime continuous cyclic voltammetric measurements. The ability of the sensor to monitor drug delivery was investigated by using a commercial micro-osmotic pump. Results show that the MWCNT/msCYP1A2-SPE sensor is capable of precisely monitoring the real-time delivery of NAP for 16 h. This work proves that the proposed electrochemical sensor might represent an innovative point-of-care solution for the personalization of drug therapies, as well as for pharmacokinetic studies in both animals and humans.

Multiplexing pH and temperature in a molecular biosensor

Robust and reliable measurements in electrochemical biosensing of molecules are crucial for personalized medicine. Electrochemical sensors based on cytochrome P450 can detect the large majority of drugs commonly used in pharmacological treatments. The same cytochrome can detect different substrates; each of them changes the electrochemical response of the enzyme in a specific manner. Our system exploits the measure of electrical potential to identify the drug type, while current measurements decode the drug concentration. Since potential and current are affected by pH and temperature, and since variations occur in the patient samples, we propose a novel design for multiplexing biosensing with pH and temperature control, which ensures more precise measurements for drugs identification and their quantification.

An Integrated Control and Readout Circuit for Implantable Multi-Target Electrochemical Biosensing

We describe an integrated biosensor capable of sensing multiple molecular targets using both cyclic voltammetry (CV) and chronoamperometry (CA). In particular, we present our custom IC to realize voltage control and current readout of the biosensors. A mixed-signal circuit block generates sub-Hertz triangular waveform for the biosensors by means of a direct-digital-synthesizer to control CV. A current to pulse-width converter is realized to output the data for CA measurement. The IC is fabricated in 0.18 μm technology. It consumes 220 μW from 1.8 V supply voltage, making it suitable for remotely-powered applications. Electrical measurements show excellent linearity in sub- μA current range. Electrochemical measurements including CA measurements of glucose and lactate and CV measurements of the anti-cancer drug Etoposide have been acquired with the fabricated IC and compared with a commercial equipment. The results obtained with the fabricated IC are in good agreement with those of the commercial equipment for both CV and CA measurements.

A Subcutaneous Biochip for Remote Monitoring of Human Metabolism: Packaging and Biocompatibility Assessment

This paper represents the extended version of the conference paper “Developing highly-integrated subcutaneous biochips for remote monitoring of human metabolism” presented at the IEEE Sensors Conference 2012, and presents data on assembly, packaging and short term in vitro and in vivo biocompatibility evaluation of a fully implantable biosensor array. The device was realized integrating three building blocks: 1) a multielectrode platform; 2) an inductive coil; and 3) an integrated circuit. The entire system measures 2.2 mm × 2.2 mm × 15 mm. Corrosion of electronic components and leaking of potentially hazardous substances in the body is prevented with a conformal coating of Parylene C, while an outer package of medical grade silicone was employed to create a soft shell suitable for implantation. Biocompatibility experiments did not show in vitro cytotoxicity in the considered period of 7 days, while comparison between 7 and 30 days in vivo implantations showed significant reduction of the inflammatory response in time, suggesting normal host recovery.

Multiplexed nanoplasmonic biosensor for one-step simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine

Development of rapid and multiplexed diagnostic tools is a top priority to address the current epidemic problem of sexually transmitted diseases. Here we introduce a novel nanoplasmonic biosensor for simultaneous detection of the two most common bacterial infections: Chlamydia trachomatis and Neisseria gonorrhoeae. Our plasmonic microarray is composed of gold nanohole sensor arrays that exhibit the extraordinary optical transmission (EOT), providing highly sensitive analysis in a label-free configuration. The integration in a microfluidic system and the precise immobilization of specific antibodies on the individual sensor arrays allow for selective detection and quantification of the bacteria in real-time. We achieved outstanding sensitivities for direct immunoassay of urine samples, with a limit of detection of 300 colony forming units (CFU)/mL for C. trachomatis and 1500CFU/mL for N. gonorrhoeae. The multiplexing capability of our biosensor was demonstrated by analyzing different urine samples spiked with either C. trachomatis or N. gonorrhoeae, and also containing both bacteria. We could successfully detect, identify and quantify the levels of the two bacteria in a one-step assay, without the need for DNA extraction or amplification techniques. This work opens up new possibilities for the implementation of point-of-care biosensors that enable fast, simple and efficient diagnosis of sexually transmitted infections.

Remote System for Monitoring Animal Models With Single-Metabolite Bio-Nano-Sensors

A novel system for remote monitoring of metabolism in an animal model is proposed in this paper. The system is obtained by integrating bio-nano-sensors to detect single-metabolites, an electrochemical front-end made with off-the-shelf components, a radio frequency communication sub-system, and an antenna of new design. The system has been calibrated and tested for continuous monitoring of four different metabolites: glucose, lactate, glutamate, and adenosine triphosphate. Tests using animal models (mice) have been conducted to investigate tissue inflammation induced by the implanted bio-nano-sensors. These tests confirm that our system is suitable and reliable for remote monitoring of single-metabolites in experiments with animal models.

New probe immobilizations by lipoate-diethalonamines or ethylene-glycol molecules for capacitance DNA chip

Label-free DNA detection is of crucial role to when developing point-of-care biochips to be used in personalized therapy. Capacitance detection is a promising technology for label-free DNA detection. However, data published in literature often show evident time drift, large standard deviation, scattered data points, and poor reproducibility. To solve these problems, alkanethiol molecules such as mercapto-hexanol are usually considered as blocking agents. The aim of the present paper is to investigate new blocking agents to further improve DNA probe surfaces. Data from AFM, SPR, florescence microscopy, and capacitance measurements are used to demonstrate the new lipoates molecules. Moreover precursor layers obtained by using Ethylene-glycol alkanethiols offer further improvements in terms of diminished detection errors. Film structure is investigated at the nano-scale to justify the detection improvements in terms of probe surface quality. This study demonstrates the superiority of lipoate and Ethylene-glycol molecules as blocking candidates when immobilizing molecular probes onto spot surfaces in label-free DNA biochip.

Developing highly-integrated subcutaneous biochips for remote monitoring of human metabolism

A highly integrated system for remote monitoring of several human metabolites is proposed. It is obtained by integrating several components, such as: bio-probes, carbon nanotubes, micro-fabricated gold-electrodes, temperature and pH sensors, extremely small CMOS IC and multilayer coil for remote powering data acquisition and transmission. The entire system has final sizes of only 2.2 mm in width and thick and it is long 1.5 cm. In this paper, results on sensors micro-fabrication, sensing tests, IC design, and power transmission are presented. These results confirmed that the proposed approach is suitable for minimally invasive monitoring of multi-metabolites in humans.